**7.1 High performance liquid chromatography**

When we draw the attention towards the working principle of HPLC, which involves the injection of small sample (Generally in μl) into the stationary phase composed of 3–5-micron tiny particles. The injected components of the sample moved through the abovementioned stationary phase with the mobile phase which is forced through high pressure by the pump.

The HPLC technique is having advantage of High speed, Efficiency, Sensitivity and vastly superior over the simple liquid chromatography. The process of separation of components of sample involves chemical and physical interactions with the stationary phase particles. The separated components are detected at the end of column by the detector in the form of the liquid chromatogram (**Figure 4**).

The HPLC can be applied for separation of non-volatile compounds like Aspirin, Ibuprofen, Acetaminophen and then for separation of salts like Sodium Chloride, Potassium Phosphate. For the separation of proteins like Egg white and Blood

**163**

**Figure 4.**

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

proteins. The HPLC can also be applied for separation of Organic Polymers, Heavy Hydrocarbons, Natural Products, Thermally unstable compounds and Enzymes [14]. For separation of many complex mixtures including biological samples high performance liquid chromatography is the best form of liquid chromatography (HPLC). HPLC is widely used for qualitative and quantitative analysis of different types of pharmaceuticals due to its sensitivity. By using HPLC, one can obtain individual sample with its role in that sample. In 20th century, the HPLC methods were appeared for assay of bulk drugs and later become a principal method of Pharmacopeia. The interaction among the solute molecules and stationary phase, decides the mode of chromatography. HPLC is more versatile technique as various modes are available. By using HPLC, the proper values of precision can obtain with excellent specificity of the methods. Though the specificity, precision and accuracy are obtained with HPLC methods, the system suitability parameters are first analyzed before analyzing these parameters. The more attention should also be providing for high accuracy, precision and specificity. By doing wide literature survey it was observed that HPLC is widely used technique among all chromatographic techniques. One of the reasons for this is detection system of HPLC which can able to detect every component of mixture. The UV detector is most widely used detector for HPLC. The Ultra-violet (UV) detector can analyze various wavelengths simultaneously by giving multiple wavelength programmers on HPLC software. Every component present in mixture which UV can detect that can be obtained by UV detector. A Photodiode array (PDA) detector is one of useful spectroscopic detector. By placing at the image plane of spectrophotometer various wavelength can be scanned simultaneously. For analysis of alcohols, sugars, carbohydrates, fatty acids and polymers, the refractive index detector is used as there is restriction for UV absorption of these compounds. The Refractive Index (RI) detector is one of the lowest sensitivity detectors but it can be applied for trace detection with low noise. For analyzing oxidizable and reducible substances, the electrochemical detector is implemented. In this detector the electrical output obtained by electron flow due to chemical reaction at electrode surface due to presence of above-mentioned compound is used for qualitative and quantitative analysis of these types of samples. Among various detectors available for HPLC, the most sensitive detector is fluorescence detector. The sensitivity of fluorescence detector is 10–1000 times more sensitive as compared with the UV detector. If sample contains any specific

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

*Flow diagram of working principle of HPLC.*

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

**Figure 4.** *Flow diagram of working principle of HPLC.*

*Analytical Chemistry - Advancement, Perspectives and Applications*

**7. Analytical techniques for method development and validation**

The various analytical techniques are available for Qualitative and Quantitative analysis, which can be used in above explained types of analytical methods. The chromatographic techniques used as a separation tool and spectroscopic techniques are used for an identification and to obtain structural information. Among all these techniques High performance liquid chromatography, High performance thin layer chromatography, Spectrophotometric techniques and Hyphenated techniques are

When we draw the attention towards the working principle of HPLC, which involves the injection of small sample (Generally in μl) into the stationary phase composed of 3–5-micron tiny particles. The injected components of the sample moved through the abovementioned stationary phase with the mobile phase which

The HPLC technique is having advantage of High speed, Efficiency, Sensitivity and vastly superior over the simple liquid chromatography. The process of separation of components of sample involves chemical and physical interactions with the stationary phase particles. The separated components are detected at the end of column by the detector in the form of the liquid chromatogram (**Figure 4**).

The HPLC can be applied for separation of non-volatile compounds like Aspirin, Ibuprofen, Acetaminophen and then for separation of salts like Sodium Chloride, Potassium Phosphate. For the separation of proteins like Egg white and Blood

**162**

explained in brief.

**Figure 3.**

**7.1 High performance liquid chromatography**

*Sequence showing development and validation of bioanalytical method.*

is forced through high pressure by the pump.

proteins. The HPLC can also be applied for separation of Organic Polymers, Heavy Hydrocarbons, Natural Products, Thermally unstable compounds and Enzymes [14].

For separation of many complex mixtures including biological samples high performance liquid chromatography is the best form of liquid chromatography (HPLC). HPLC is widely used for qualitative and quantitative analysis of different types of pharmaceuticals due to its sensitivity. By using HPLC, one can obtain individual sample with its role in that sample. In 20th century, the HPLC methods were appeared for assay of bulk drugs and later become a principal method of Pharmacopeia. The interaction among the solute molecules and stationary phase, decides the mode of chromatography. HPLC is more versatile technique as various modes are available. By using HPLC, the proper values of precision can obtain with excellent specificity of the methods. Though the specificity, precision and accuracy are obtained with HPLC methods, the system suitability parameters are first analyzed before analyzing these parameters. The more attention should also be providing for high accuracy, precision and specificity. By doing wide literature survey it was observed that HPLC is widely used technique among all chromatographic techniques. One of the reasons for this is detection system of HPLC which can able to detect every component of mixture. The UV detector is most widely used detector for HPLC. The Ultra-violet (UV) detector can analyze various wavelengths simultaneously by giving multiple wavelength programmers on HPLC software. Every component present in mixture which UV can detect that can be obtained by UV detector. A Photodiode array (PDA) detector is one of useful spectroscopic detector. By placing at the image plane of spectrophotometer various wavelength can be scanned simultaneously. For analysis of alcohols, sugars, carbohydrates, fatty acids and polymers, the refractive index detector is used as there is restriction for UV absorption of these compounds. The Refractive Index (RI) detector is one of the lowest sensitivity detectors but it can be applied for trace detection with low noise. For analyzing oxidizable and reducible substances, the electrochemical detector is implemented. In this detector the electrical output obtained by electron flow due to chemical reaction at electrode surface due to presence of above-mentioned compound is used for qualitative and quantitative analysis of these types of samples. Among various detectors available for HPLC, the most sensitive detector is fluorescence detector. The sensitivity of fluorescence detector is 10–1000 times more sensitive as compared with the UV detector. If sample contains any specific

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