Role of HPLC in Cleaning Validation of Laboratory Glassware

Cleaned and validated glassware ready for use

HPLC has made significant contributions in detection and quantitative estimation of organic compounds. In pharmaceutical analysis HPLC has been extensively used in assay of the active ingredient as well as related impurities which are present in formulations in very low concentrations. Estimation of related substances was not possible using other conventional analysis techniques. This unique ability of HPLC has been successfully exploited for validation of glassware cleaning before it can be put to use for making sensitive analytical determinations.

It is clear that glassware should be cleaned to your complete satisfaction so that you are confident of the results communicated by you. In case you lack confidence then you should be prepared for criticisms on your reports. Cleaning of laboratory glassware suggests cleaning practices for different types of glassware based on the analysis requirements.

Next the question will arise if you can rely on adequacy of your cleaning or further cleaning would be necessary. In other words cleaned glassware will require validation for cleanliness before use. Cleaning validation is a set of practices laid down to ensure the effectiveness and consistency of the cleaning method for removal of residues from earlier analysis, contaminants or even from the detergents used for cleaning. 

Laboratory glassware cleaning involves repetitive rinses with deionised water or an organic solvent followed by drying in hot air oven. It is important to use water or solvent of high purity grade so that it does not contribute to the contamination of the glassware. During analysis using sophisticated analytical techniques such as HPLC you can come across a set of peaks (referred to as ghost peaks) whose presence cannot be explained. The first thing that can come to your mind could be that such peaks result from impurities present in available standards, blank solutions, reagents or even unclean samples. Such sources of contamination can be investigated independently but due consideration should be given to inadequate cleaning of the glassware before putting it to use.

The residues remaining after cleaning and levels of detergents used in the cleaning process or trace impurities present in de-ionized water or organic solvents necessitates use of a sensitive analytical technique. HPLC is a good choice as it offers a large number of possibilities due to selection from a range of columns and detectors. Generally HPLC systems with a UV detector, photodiode array detector and a C-18 column in reverse phase mode is adequate for the purpose. HPLC offers high sensitivity and detection limits besides providing advanced automation features that can contribute to large scale studies on cleanliness validation of volumetric glassware items as per requirements mandated by the standard.

Requirement based cleaning of Laboratory Glassware

Laboratory Glassware

A laboratory makes use of several glassware items such as volumetric flasks, burettes, pipettes, beakers, watch glasses, specific gravity bottles,vials,petri dishes, graduated cylinders, etc. Such items are not disposables and are used repeatedly. However, the quality of analytical results is dependent to a large extent on their cleanliness and freedom from contamination.

Laboratory glassware cleaning practices covered briefly the adopted glassware cleaning practices.The present article offers suggestions for cleaning of different types of glassware and their cleaning practices after handling of different classes of chemicals.

Cleaning of different categories of glassware

Glassware for laboratory applications should be inspected carefully before use. They should be free from spots and visibly clean, free from chemical contamination and also free from bacteria,ie,sterile before being used in microbiological applications. A simple method adopted for checking freedom from oil or grease contamination is uniform wetting by a stream of distilled water. Presence of grease or oil will prevent uniform wetting and result in formation of minute droplets.

Volumetric Flasks

After use immediately soak in warm soap water. Clean any stubborn deposits with a soft brush. Rinse with tap water followed by 3 – 4 rinses with deionised water. Hot air oven drying should be avoided as this can affect the volumetric calibration of glassware. In case glassware is urgently required rinse 2- 3 times with acetone and apply vacuum to remove the residual acetone. Air blowing should be avoided as it can contaminate the cleaned flask.

Burettes

Wash with soap water. Use long handle soft brush if necessary. and finally rinse 2 – 3 times with deionised water. Remove the joint and boil in a weak solution of sodium carbonate to remove grease. Apply a thin film of grease after cleaning and re-insert into the burette.

Pipettes

Insert pipettes gently into a jar filled with tap water for some time. After removal dip in a jar of dissolved detergent or chromic acid and after soaking for sufficient time (several hours) run deionised water both outside and inside. Wipe outside with lint free tissue. Allow to dry.

Culture tubes and Petri dishes

Microbiological studies required use of culture tubes and Petri dishes. Sterilize before cleaning in the autoclave for 20 – 30 minutes at 121 degrees centigrade and 15 psi pressure. Empty the contents and adopt general cleaning practices as for other glassware.

Cleaning after use of different types of chemicals

Commonly you would encounter aqueous or non-aqueous samples in laboratory analysis.After using aqueous solutions you should wash glassware with tap water followed by rinsing 3 to 4 times with deionised water.In case of non-aqueous samples rinsing with ethanol or acetone is sufficient followed by 3 – 4 rinses with deionised water.

Strong acids and bases

Wear gloves and safety glasses for the cleaning operation. Rinse with several volumes of tap water in a fume hood followed by 3 to 4 rinses with deionised water

Weak acids and bases

Glassware used for handling weak acids or bases or dilute solutions of strong acids or bases can be rinsed with tap water followed by 3-4 rinses with deionised water

Autoclaving

Steam autoclaving for sterilization requires essential precautions:

• Remove closure caps, if any, before autoclaving
• Inspect glassware carefully for cracks. Autoclave crack free glassware only as cracked or chipped glassware can break during the process of sterilization.
• Allow the glassware to attain room temperature after autoclaving so as to avoid handling injuries or breakages.
• Only borosilicate glass items should be autoclaved. Bottles which are usually made from soda lime glass do not withstand the autoclave temperatures.
• The autoclave should be provided with auto cut-off in case there is accidental increase in temperature or pressure.

Take adequate precautions when cleaning laboratory glassware. Protection of eyes, hands and dress is essential so as to avoid cut injuries due to breakages, soap solution entry into eyes and causing irritation. Other harsh cleaning agents can cause skin burns or even produce toxicity through skin penetration.

Laboratory Glassware Cleaning Practices

Use of clean laboratory glassware is critical for high accuracy and precision of your results. Cleanliness of glassware up to the required degree prior to start of analytical work is often underestimated and some analysts feel the job can be entrusted to laboratory support staff. A dedicated analyst on the other hand is never ashamed of washing laboratory glassware to his or her own satisfaction.

Breakages can be frustrating and lead to loss of valuable time. A few precautions taken can avoid such embarrassing situations

• Prevent breakage by exercising care by avoiding hitting of burettes, pipettes or measuring cylinder tips to tap or washbasin walls
• Measuring cylinders, Centrifuge tubes, flasks, etc should be dried on wooden or plastic pegs mouth downwards
• Only soft brushes should be used if necessary. Never clean absorption cells with brushes as scratches will lead to wrong absorbance readings and result in optical mismatch of the cell pair
• Volumetric flask should never be dried in the drying oven with temperatures exceeding 65°C. Always un stopper cover before placing in the oven

Cleaning Agents

Used laboratory glassware should be soaked in water even if immediate cleaning is not possible. Most commonly available detergents can be used for subsequent cleaning. An oxidizing agent such as Potassium dichromate in sulphuric acid is a good choice. The solution becomes green on continued use due to loss of its oxidizing power and should be discarded. A mixture of 3N HCL and 1 volume of methyl alcohol is useful for cleaning of stains in absorption cells. This mixture should not be shaken in closed vessels due to generation of heat.

Grease can be removed from the joints and stoppers by boiling in a weak solution of sodium bicarbonate and subsequently soaked in a solution of potassium dichromate or acetone prior to rinsing with distilled water.

New laboratory glassware is slightly alkaline. Soak in 1% HNO3 or HCl solution for about an hour and then wash with distilled water before drying.

Laboratory Glassware washing machines

Labware Washing Machines

Laboratory glassware washing machines provide superior cleaning and convenience. Valuable laboratory time is saved when bulk washing is necessary. Such machines can accommodate a wide range of glassware shapes and sizes with adjustable racks to accommodate wide mouth glassware (beakers, conical flasks, graduated cylinders, etc) as well as narrow mouth glassware (volumetric flasks). Cycle operations permit wash and dry operations between rinses. Built-in heating of wash water and forced air drying are common features provided by most suppliers.