Poly (lactide-co-glycolide acid) ( PLGA) is an extraordinary well-described polymer and has excellent pharmaceutical properties like high biocompatibility and good biodegradability. Hence, it is one of the most used materials for drug delivery and biomedical systems, also being present in several US Food and Drug Administration approved carrier systems and therapeutic devices. For both applications, the quantification of polymer is important. During the development of the production process, parameters like yield or loading efficacy are essential to be determined. Although PLGA is a well-defined biomaterial, it still lacks a sensitive and convenient quantification approach for PLGA-based systems. Thus, we present a new method for fast and precise quantification of PLGA by HPLC-SEC. The method includes a shorter run time of 20 minutes with a size exclusion column of 300mm x 8.0mm diameter, tetrahydrofuran as mobile phase and diluent, the detection was carried out using the refractive index detector. The developed method has a detection limit of 0.1 ppm, enabling the quantification of low amounts of PLGA. Compared to existing approaches, like gravimetric or nuclear magnetic resonance measurements, which are tedious or expensive, the developed method is fast, ideal for routine screening and it is selective since no interference. The developed method is validated in terms of selectivity, precision, linearity, accuracy and solution stability. Due to the high sensitivity and rapidity of the method, it is suitable for both, laboratory and industrial use &nbsp;&nbsp

D. Kayande, Deepak

Deshmukh, Parag

Farooqui, Mazahar

Shripad

English

PURKH (E-Journals)

To Chemistry Journal Vol 4 (2019) ISSN: 2581-7507                                                       http://purkh.com/index.php/tochem 
18 
High Performance Liquid Chromatography-Size Exclusion Chromatography (Hplc-Sec) As an Efficient 
Tool for The Quantification of Polymers. 
Deepak D. Kayande1* Shripad Deshpande1, Mazahar Farooqui2, Parag Deshmukh3 
1 S.B.E.S. College of science, Aurangabad, Maharashtra, India-431001. 
2 Maulana Azad College, Aurangabad, Maharashtra India-431004. 
3 ICICI knowledge park Shameerpet, Hyderbad, 500078 India. 
Drkayande@gmail.com 
Abstract 
Poly (lactide-co-glycolide acid) ( PLGA) is an extraordinary well-described polymer and has excellent 
pharmaceutical properties like high biocompatibility and good biodegradability. Hence, it is one of the most 
used materials for drug delivery and biomedical systems, also being present in several US Food and Drug 
Administration approved carrier systems and therapeutic devices. For both applications, the quantification of 
polymer is important. During the development of the production process, parameters like yield or loading 
efficacy are essential to be determined. Although PLGA is a well-defined biomaterial, it still lacks a sensitive and 
convenient quantification approach for PLGA-based systems. Thus, we present a new method for fast and 
precise quantification of PLGA by HPLC-SEC. The method includes a shorter run time of 20 minutes with a size 
exclusion column of 300mm x 8.0mm diameter, tetrahydrofuran as mobile phase and diluent, the detection was 
carried out using the refractive index detector. The developed method has a detection limit of 0.1 ppm, enabling 
the quantification of low amounts of PLGA. Compared to existing approaches, like gravimetric or nuclear  
magnetic resonance measurements, which are tedious or expensive, the developed method is fast, ideal for  
routine screening and it is selective since no interference. The developed method is validated in terms of 
selectivity, precision, linearity, accuracy and solution stability. Due to the high sensitivity and rapidity of the 
method, it is suitable for both, laboratory and industrial use.      
Keywords: Poly (Lactide-Co-Glycolide Acid); PLGA; Quantification; HPLC-SEC, Refractive Index. 
Introduction 
Since the introduction of Poly (lactic-co-glycolic acid) (PLGA) as bioresorbable fiber material in 1960, PLGA 
became one of the most used polyesters for the development of drug delivery and biomedical systems [1-3]. 
PLGA is a copolymer of lactic acid (LA) and glycolic acid (GA), with varying ratios, and belongs to a group of 
highly biodegradable and biocompatible polymers. The US Food and Drug Administration (FDA) approved 
PLA/PLGA based drug products that are currently available, and due to its enormous popularity, it is one of the 
best-defined biomaterials available for drug delivery. In aqueous medium the ester backbone of PLGA 
undergoes slow hydrolysis. The polymer degrades into the monomers LA and GA both being entirely eliminated 
from the body. Lactic acid converts to pyruvate, which degrades into water and carbon dioxide via the Krebs 
cycle [4]. Glycolic acid will be either excreted directly via urine or will be oxidized into glyoxylate, which will be 
converted to glycine, serine, and pyruvate [5]. For particle engineering, the tenability of the material is essential. 
Therefore, characteristics like surface modifications or adjustment of physicochemical properties, and the 
biodegradation rate of the material are crucial. Several protocols for the preparation and modification of micro 
and nanoscale PLGA systems have been established [6-10]. Depending on the manufacturing method and the 
operating parameters, the yield of the method and the loading efficiency of nano and microparticles can vary 
drastically, influencing the therapeutic use [11-12]. Despite the high interest in PLGA, a convenient method for 
the quantification of the polymer matrix is still lacking. Usually, the PLGA system is estimated by gravimetric 
methods after sample lyophilization or, for increasing the sensitivity, by more complex methods like mass 
spectrometry, nuclear magnetic resonance or other analysis methods [13-17]. However, these approaches suffer 
To Chemistry Journal Vol 4 (2019) ISSN: 2581-7507                                                       http://purkh.com/index.php/tochem 
19 
partly from sensitivity, as the pure polymer matrix cannot be quantified selectively or the required equipment 
for improving the analysis sensitivity is expensive and the quantification time consuming. In general, PLGA based 
drug delivery or biomedical systems are a complex mixture, composed of several molecules like loaded drugs 
and excipients. For estimating the yield of the manufacturing techniques, adjusting the concentration of assays, 
determining the loading efficiency and therapeutic dose of a drug or for fast quality control of the product, 
knowledge about the exact PLGA amount in the formulation is essential. This study presents a rapid and precise 
method for the selective quantification of PLGA. For the development, the technological requirements were kept 
as low as possible without losing selectivity and sensitivity. PLGA soaked and dissolved in tetrahydrofuran 
solvent. This PLGA solution is quantified against the different molecular weight polystyrene standards in a high-
performance liquid chromatography-size exclusion chromatography (HPLC-SEC). The chromatographic 
conditions mainly content the organic size exclusion column with a dimension of 300mm x 8.0mm, 
tetrahydrofuran as a diluent and mobile phase, column temperature of 40°C, sample temperature of 10°C with 
a run time of 20 minutes. The detection was carried out on a refractive index detector with sampling rate 1.0, 
sensitivity 16 and detector temperature of 35°C. This method even allows the quantification of low amounts of 
PLGA. Hence the developed method is suited for various production phases.    
Materials and Methods 
Poly (lactic-co-glycolic acid) with a glycolic/lactic acid ratio of 50:50 and 75:25 was purchased from Evonik 
Nutrition, Expansorb, and Picas. The reference standards with different molecular weights are purchased from 
Waters Corporation. The solvents acetonitrile, methanol purchased from Merck limited. Stabilized 
tetrahydrofuran was purchased from J.T. Baker. 
Preparation of standards for determination of molecular weight 
Accurate weight of each Polystyrene standard taken in a dry volumetric flask to obtain a 1000ppm of 
concentration. The polystyrene standards in this study having theoretical molecular weights of 10700, 19900, 
42400,116000 and 264000. All weighed standards are kept for soaking for 30 minutes of time. Each individual 
polymer was filled in the HPLC vials for analysis. 
Quantitative analysis of Polystyrene standards and PLGA    
For the quantification of PLGA, an HPLC system (Waters Corporation), equipped with a quaternary pump and a 
refractive index detector used. Furthermore, the system used an autosampler with 10°C, a column oven 40°C, 
and a column Shodex HT 804 (300mm x8.0 mm and 13 µm). Stabilized Tetrahydrofuran solvent is used as a 
mobile phase and diluent. The degasser mode was kept off in HPLC.  The column was saturating with 
tetrahydrofuran for overnight with a flow of 0.2mL per minutes. The 0.7 mL per minute flow is given in method 
with the injection volume of 10µL. For data analysis, the chromatography software Empower 3.0 was used. The 
retention time and observed molecular weights with a correlation of more than 0.99 were considered as 
authentic. With the help of this calibration curve standards and retention time of particular PLGA obtained in 
the sample, the Molecular weight of that particular sample is determined. This optimized method is validated 
for specificity, linearity, precision, accuracy and solution stability.      
Results and discussion 
Various parameters were analyzed and optimized for the sensitive quantification of the method. 
Specificity 
The specificity of the method is carried out by injecting the blank tetrahydrofuran in the set method and 
observed that no interfering peak is observed from the blank at the retention of Polystyrene standard and PLGA 
sample retention. 
To Chemistry Journal Vol 4 (2019) ISSN: 2581-7507                                                       http://purkh.com/index.php/tochem 
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Linearity 
The linearity of the method is proposed by injecting five different molecular weight standards and observing its 
correlation coefficient. Table 1 shows the actual molecular standard weight, observed retention time, observed 
molecular weight and correlation coefficient obtained through software. Similarly Figure 1 shows the calibration 
plot of time versus molecular weight.  
Figure 1: Standard calibration plot 
Table 1: Molecular weight calibration table 
Standard 
details 
Molecular weight 
(Daltons) 
Retention Time 
(min) 
Calculated weight 
(Daltons) 
R 2 
1 264000 9.200 261666 
0.999561 
2 116000 9.958 118600 
3 42400 11.385 40961 
4 19900 12.363 20559 
5 10700 13.114 10579 
Precision 
System suitability and method precision carried out by injecting known molecular weight standards and PLGA 
containing samples six times. Below mentioned figures 2-6 shows the suitability of the system. The results 
mentioned in table 2 show the preciseness of the method. 
 
 
To Chemistry Journal Vol 4 (2019) ISSN: 2581-7507                                                       http://purkh.com/index.php/tochem 
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Figure 2: Molecular weight calibrant 10700 
 
Figure 3: Molecular weight calibrant 19900 
Figure 4: Molecular weight calibrant 42400 
To Chemistry Journal Vol 4 (2019) ISSN: 2581-7507                                                       http://purkh.com/index.php/tochem 
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Figure 5: Molecular weight calibrant 116000 
Figure 6: Molecular weight calibrant 264000 
Table 2: Method precision results 
Sample no. Mn Mp Mz Polydispersity Mw 
1 65630 102184 163276 1.59 104643 
2 67642 105932 167257 1.58 106942 
3 63994 102958 195173 1.74 111457 
4 66545 104565 175225 1.65 108850 
5 67098 103960 180850 1.58 117810 
6 66110 104668 185590 1.67 108758 
Mean 109743 
SD 4552.7 
%RSD 4.15 
Accuracy 
Accuracy of the method is determined by the percentage relative difference between the theoretical molecular  
weight of standard and calculated molecular weight of standard at the initial 24 Hrs. of the time interval. 
 
To Chemistry Journal Vol 4 (2019) ISSN: 2581-7507                                                       http://purkh.com/index.php/tochem 
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Observations for known standard solution (Initial) as broad unknown:  
Table 3: Standard solution details at initial 
Standard name Theoretical molecular 
weight of standard 
Calculated molecular 
weight of standard 
% Relative difference 
1.07 x 104 10700 10695 0.05 
1.99 x 104 19900 20646 -3.75 
4.24 x 104 42400 41312 2.57 
1.16 x 105 116000 118937 -2.53 
2.64 x 105 264000 262571 0.54 
Observations for known standard solution (24 Hrs) as broad unknown:  
Table 4: Standard solution details after 24 Hrs. 
Standard name Theoretical molecular 
weight of standard 
Calculated molecular 
weight of standard 
% Relative difference 
1.07 x 104 10700 10772 -0.67 
1.99 x 104 19900 20704 -4.04 
4.24 x 104 42400 41414 2.33 
1.16 x 105 116000 116054 -0.05 
2.64 x 105 264000 254862 3.46 
Conclusion 
Although PLGA is a well-described and one of the most used polymers in many research fields, it still lacks a 
simple, fast and reliable quantification method. Therefore, a new HPLC-SEC method for the quantification of 
PLGA was developed. The presented study is first fully validated; the described method combines low 
instrumental requirements with short preparation time and high precision and selectivity for polymers. The 
validated parameters show a good range for polymer determination with high precision. Therefore, the present 
method can be considered as simple, fast, and easy to apply, making it very suitable for routine analysis in 
quality control of PLGA based systems. The presented method can be used for a dual quantification of the 
polymer matrix and the encapsulated drug within a single run. The reported method was very suitable for routine 
analysis in quality control of PLGA based systems with promising future application features.  
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High Performance Liquid Chromatography-Size Exclusion Chromatography (Hplc-Sec) As an Efficient Tool for The Quantification of Polymers

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High Performance Liquid Chromatography-Size Exclusion Chromatography (Hplc-Sec) As an Efficient Tool for The Quantification of Polymers

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