Your Question :-

How to analyse UV inactive compound on HPLC using UV Detector?

In my research field I got UV inactive compound having one Chiral Center I want to separate the enantiomers on HPLC having UV detector So what can i do to get UV response on HPLC. ?

Answer - 

I suggest to complex the compound with some kind of regents to give a color so you can detect in visible range. For the enantiomer you can modify a mobile phase containing 5% beta-cyclodextrine to enhance selectivity towards enentiomers.Derivatization is one way, but more laborious and expensive. You can try the more straightforward approach of indirect UV detection.  The detection of a UV-Vis-transparent analyte is accomplished by adding light-absorbing species into the mobile phase. The presence of the analyte is monitored by measuring a decrease or The only way you could analyze a UV inactive compound on a UV detector is to derivatize it to add a UV active functionality, such as a phenyl ring.  Otherwise you can't use UV on the compounds in their native state since there is nothing for the UV detector to detect. If you are doing this as a preparative LC than derivatization wouldn't be an option.  Your only recourse would be to use another type of detector.

 Your Question :-

    Can Chloroform degrade C18 reverse phase Silica?

Answer - Chloroform will not damage your C18 RP column. I was hesitant before using it the first time, so I used a really old column that had been used with a lot of lipid-containing samples. The improvement after a {20% Ethanol or Methanol:water(ultra-pure) > 100% Methanol > 100% Acetonitrile > 100% Chloroform > 100% Acetonitrile > 100% Methanol > 20% Methanol:ultra-pure water} progression was very dramatic, restoring the column's theoretical plate number to close to that when it was new. This suggested that a lot of lipid had been accumulating on my column, impairing its performance and maybe introducing a 'lipid partition' into my separations. I have repeated this 'column-stripping' procedure several times since, on old columns that were losing resolution, and it does usually help, depending on the history of the column, and whether it has been used and stored within the limits set by the manufacturer.

I agree, however, that chloroform would not be appropriate in a mobile phase solvent for a Reverse-Phase column.

                                                                                                                                Thank you

  Note - Drop your Question in Question box and also give your feedback about contents.

HPLCWOOD: CARRYOVER TROUBLE in HPLC and Action Steps

HPLCWOOD: CARRYOVER TROUBLE in HPLC and Action Steps:                         HPLC Carry over and its Solution to prevent Carryover Discription   Carryover is recognized as the presence of a sma...

DATA INTEGRITY AND ALCOA +

 

                        DATA INTEGRITY AND ALCOA + 

                        

Your  question ---

Q: I am familiar with the term ALCOA as it relates to data integrity, but lately, I have heard people refer to ALCOA+. Can you explain what impact this new acronym has on my company’s data integrity program?

ANSWER : -  Before we get into the reason for the additions to ALCOA, referred to as ALCOA+, we should review what both of the acronyms mean. The acronym ALCOA requires data be attributable, legible, contemporaneous, original, and accurate. The acronym ALCOA+ adds the concepts that, in addition to ALCOA, data also needs to be complete, consistent, enduring, and available. It is important to understand what each element of ALCOA and ALCOA+ mean in order to apply the concepts appropriately with respect to a company’s records. The following are some general definitions, paraphrased from the Pharmaceutical Inspection Co-operation Scheme (PIC/S) (1), that can be used for understanding the elements of ALCOA and ALCOA+:

• Attributable: The data generated or collected must be traceable back to the individual who generated the information.

• Legible: The data recorded must be readable and permanent.

• Contemporaneous: The results, measurements, etc. must be recorded at the time the work is performed.

• Original: Original or source data are the record, report, notebook etc. where the data point was initially recorded.

• Accurate: The data recorded must be complete, consistent, truthful, and representative of facts.

• Complete: Information that is critical to recreating and understanding an event. This would include any repeat or reanalysis performed on a laboratory test sample.

• Consistent: The data are presented, recorded, dated, or time-stamped in the expected and defined sequence.

• Enduring: The data or information must be maintained, intact, and accessible throughout their defined retention period.

• Available: The data or information must be able to be accessed at any time during the defined retention period.

Data integrity has always concerned regulatory authorities, but it is important to understand what is prompting the renewed discussion of ALCOA and the introduction of ALCOA+ when discussing data integrity issues. Many of the concepts for ALCOA have been captured in the regulations as far back as 1978. Since that time, the industry has changed dramatically. The generic-drug industry has grown and in the United States alone accounts for more than 80% of the prescriptions written today (2). Coupled with the emergence of biosimilars, virtual companies, contract manufacturing organizations, rapid advances in automation and information technology, and the globalization of the industry have resulted in reinterpretation of the attributes associated with maintaining the integrity of data throughout the product lifecycle, whether those data are generated from electronic, paper-based, or hybrid systems. In addition, there has been an increase in citations internationally by the FDA, European Medicines Agency (EMA), Medicines and Healthcare products Regulatory Agency (MHRA), World Health Organization (WHO), and other health authorities. These changes and increased violations have brought about a resurgence and need to reeducate the industry on the basic principles and concepts regarding the proper control of data used to support the quality and safety of medicines.

The ALCOA acronym has been used since the 1990s; however, the requirements governing data elements have been in regulations for a much longer period of time. EudraLex chapter 4 states, “Suitable controls should be implemented to ensure the accuracy, integrity, availability, and legibility of documents. Instruction documents should be free from errors and available in writing” (3). The US Code of Federal Regulations (CFR) refers to these elements in various sections of the regulations (4–8). An example for language speaking to the element of attributable is in 21 CFR 211.194(a)(7), which states, “The initials or signature of the person who performs each test and the date(s) the tests were performed.” Language in 21 CFR 58.130 (e) addresses the elements of contemporaneous and legible by stating, “All data generated during the conduct of a nonclinical laboratory study, except those that are generated by automated data collection systems, shall be recorded directly, promptly, and legibly in ink. All data entries shall be dated on the date of entry and signed or initialed by the person entering the data.”

Recent documents issued by WHO (9) and the PIC/S (1) have added to the original ALCOA attributes as indicated above. The PIC/S document actually states, “Some key concepts of GDocPs are summarized by the acronym ALCOA: Attributable, Legible, Contemporaneous, Original, and Accurate. The following attributes can be added to the list: Complete, Consistent, Enduring, and Available. Together, these expectations ensure that events are properly documented and the data can be used to support informed decisions.” WHO refers to ALCOA+ in the title of Appendix 1 to their 2018 document. The last two documents also address the concept of quality culture (10). The impact to your organization is that the quality culture must ensure that data supporting the quality and safety of your product must now meet the ALCOA+ elements in order to avoid regulatory citations for data integrity issues.

References

1. PIC/S, Draft Guidance: Good Practices for Data Management and Integrity in Regulated GMP/GDP Environments (PIC/S, August 2016).
2. statista.com 2019.
3. EC, EudraLex,The Rules Governing Medicinal Products in the European Union, Volume 4, Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use, Chapter 4.
4.  21 CFR 11: Electronic Records; Electronic Signatures
5.  21 CFR 58: Good Laboratory Practice for Nonclinical Laboratory Studies
6.  21 CFR 211: Current Good Manufacturing Practice for Finished Pharmaceuticals
7.  21 CFR 212.50: Current Good Manufacturing for Positron Emission Tomography Drugs
8.  21 CFR 820: Quality System Regulation
9.  WHO, Annex 5, Guidance on Good Data and Record Management Practices (WHO, June 2016).
10. S. Schniepp, Pharmaceutical Technology 42 (10) 2018. 

NATURE OF A GOOD MOBILE PHASE

 

                            NATURE OF A GOOD MOBILE PHASE

ABOUT MOBILE PHASE

Mobile phase has been rightly termed as the lifeline of the HPLC system. It plays the important role of transport of the sample through the separation column and subsequently to the detector for identification of the separated components.

Mobile phase is seldom a single solvent. It consists of combination of water with organic solvents, aqueous buffers with polar solvents or mixtures of organic solvents in desired proportions. The two operational modes commonly used are:

Isocratic mode – in the Isocratic mode the composition of the mobile phase remains constant throughout the analytical run ,i.e, the proportion of solvents in the mixture is pre-decided and remains unchanged during analysis

Gradient Elution – the composition is varied by the software through the analytical run in a predetermined mode and at the end of the run the proportion of solvents is different from the initial proportion.

The objective of using different solvent mixtures is to achieve the desired polarity of mobile phase for complete miscibility of the sample and control interaction of sample components with the stationary phase to achieve the desired degree of resolution of separated component peaks in the shortest possible time.

In order to meet the required objectives the mobile phase should have the following essential features:

• The sample should be fully soluble in the mobile phase. Any insolubility will result in flow restrictions. Always check sample solubility in mobile phase before injection into the system.
• Mobile phase components should be non-hazardous and non-toxic. They should not pose any health hazard to the operator.
• Mobile phase shouId be inert towards sample constituents and the stationary phase. Any reactions can lead to formation of insoluble suspensions which can result in column blockages.
• The mobile phase should not give its own response on passing through the detector. In other words the detector signals should reflect only the response of the sample constituents. This, however, is not applicable in bulk property detectors such as refractive index detector which respond to overall changes in refractive index of the mobile phase containing the eluting compound.
• The mobile phase should be affordable and the proportion of solvents used should make the analysis economically viable.

It is important to mix the solvents of specified purity and from same source to get consistency of results and also to adopt the same off-line mixing technique to avoid errors due to heat of mixing errors. In case buffers are to be used always adjust the pH of the aqueous phase prior to making up the final volume with the organic phase.

HPLC technique holds great promise for applications in diverse fields. Control of operational conditions including consistency of mobile phase will ensure repeatability of results.