**7.2 High performance thin layer chromatography (HPTLC)**

The advancement of Thin layer chromatography (TLC) is the HPTLC that is, High performance thin layer chromatography which an instrumental semi or automatic form of TLC. It is fast working, sensitive and can be able to analyze wide range of samples.

The HPTLC is advantageous to handle a sample with short analysis time for analysis of even complex samples including crude drugs. As automation with the instrument it can be able to analyze entire chromatogram with many parameters without any interruption. The samples can be analyzed simultaneously or independently with standard that shows the reliability of technique. The HPTLC is equipped with high performance adsorbent layers having refined uniform particles, approx. 5 microns in diameter. All processes of experimentation including method development, optimization of various parameters and documentation are performed with standardized methods. The HPTLC can be applied for both qualitative and quantitative analysis of mixtures, as the technique is automated, the quantitative mode is more optimized as compared with the TLC. Also, it can be used for the assay of the compound (**Figure 5**).

The advantages of the HPTLC are as follows:


**165**

electronic image [16].

**Figure 5.**

**7.3 UV-spectrophotometric methods**

*Diagram showing components of HPTLC.*

According to Beer-Lambert law,

where A = Absorbance. I0 = intensity of incident light. I = intensity of emergent light.

ɛ = molar absorptivity.

*Analytical, Bioanalytical, Stability-Indicating Methods: Key Part of Regulatory Submissions*

Development of plate and evaluation of plate are separate processes therefore

The important advantages of the HPTLC are fast, inexpensive method of analysis. It can prove over high-performance liquid column chromatography when it is performed by skilled person for quantitative analysis. The qualitative and quantitative analysis by the HPTLC with automated sample application and densitometric scanning shows very sensitive and reliable results. The HPTLC has important advantage of providing chromatographic fingerprints which can be stored as an

For the quantification of components present in solution, the UV absorption

A = log I / I = .c.l <sup>0</sup> ε

spectroscopy works on the principle of Beer-Lambert law [17] (**Figure 6**).

both can perform as per time available differently [15].

*DOI: http://dx.doi.org/10.5772/intechopen.93566*

*Analytical, Bioanalytical, Stability-Indicating Methods: Key Part of Regulatory Submissions DOI: http://dx.doi.org/10.5772/intechopen.93566*

#### **Figure 5.** *Diagram showing components of HPTLC.*

*Analytical Chemistry - Advancement, Perspectives and Applications*

(LC-MS-MS) is also available for above mentioned work [15].

**7.2 High performance thin layer chromatography (HPTLC)**

The advantages of the HPTLC are as follows:

• Colored samples can be easily separated

• Two-dimensional mode is possible

different light absorption characteristics.

The advancement of Thin layer chromatography (TLC) is the HPTLC that is, High performance thin layer chromatography which an instrumental semi or automatic form of TLC. It is fast working, sensitive and can be able to analyze wide

The HPTLC is advantageous to handle a sample with short analysis time for analysis of even complex samples including crude drugs. As automation with the instrument it can be able to analyze entire chromatogram with many parameters without any interruption. The samples can be analyzed simultaneously or independently with standard that shows the reliability of technique. The HPTLC is equipped with high performance adsorbent layers having refined uniform particles, approx. 5 microns in diameter. All processes of experimentation including method development, optimization of various parameters and documentation are performed with standardized methods. The HPTLC can be applied for both qualitative and quantitative analysis of mixtures, as the technique is automated, the quantitative mode is more optimized as compared with the TLC. Also, it can be used for the assay of the compound (**Figure 5**).

• Many samples can be assessed easily on single plate which reduces the cost and

• Visualizing agent used which are Specific and Sensitive for detection purpose

• Other evaluation technique can be implemented for different samples with

• Radio labeled compounds can be monitored and microbial activity can be

• No regeneration and cleanup are required as the technique is disposable.

fluorescent compound, then it can be easily detected by this detector. For estimation of pharmaceuticals especially fluorescence detector is applied. As most of pharmaceuticals are polar in nature, there analyses are carried out as reverse phase HPLC. In recent years most of the researchers used reverse phase chromatography with UV detection, due to that the results are obtaining with best reliability, analysis, repeatability and sensitivity. Generally, in pharmaceutical industry Octadecyl silyl (ODS) C18 is mostly used stationary phase. Many drugs can be easily obtained in pharmaceutical formulations and biological fluids by using HPLC. Nowadays, HPLC is one of the important tools for solving many problems in pharmaceutical industry. There are certain limitations to HPLC that high price of column, HPLC grade solvents and it is difficult to obtain long term reproducibility due to nature of column packings. The Liquid Chromatography-Mass spectrometry (LC-MS) is wide choice for quality control and quality assurance in various stages in pharmaceutical industry. The LC-MS can be easily applied for assay of many drugs and pharmaceuticals also applied for analysis of impurities and degradation products. The most hyphenated technique like Liquid Chromatography-Mass spectrometry-Mass spectrometry

**164**

range of samples.

time.

assessed.

Development of plate and evaluation of plate are separate processes therefore both can perform as per time available differently [15].

The important advantages of the HPTLC are fast, inexpensive method of analysis. It can prove over high-performance liquid column chromatography when it is performed by skilled person for quantitative analysis. The qualitative and quantitative analysis by the HPTLC with automated sample application and densitometric scanning shows very sensitive and reliable results. The HPTLC has important advantage of providing chromatographic fingerprints which can be stored as an electronic image [16].
