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Why is Acetonitrile Used in HPLC? | Greyhound Chromatography

Why is Acetonitrile Used in HPLC?

Acetonitrile together with Methanol are the most commonly used solvents for HPLC and UHPLC Chromatography processes because most substances are easily soluble in both solvents, but not saturated hydrocarbons.  Acetonitrile has a low wavelength which means that the solvent will elute first from the column, therefore pressure is reduced on the column.  Lower pressure in the column will have the long term effect of prolonging the life of the column, pressure within the column varies depending on the solvent used and the mixture ratio between the solvent and the sample.  An Acetonitrile based solution generally has a higher elution strength against the same strength solution using Methanol. Some scientists report that the same retention times can be achieved using acetonitrile with less than half the ratio of methanol.  Operator errors can have a significant impact of reproduceable results.  

 

Acetonitrile produces less pressure than Methanol, however the choice between using Acetonitrile against Methanol should not be made on price, (Acetonitrile is generally more expensive than Methanol), choice should be made on performance for your particular instrument and the effect of the chromatography process on the durability of the column.  High grade Acetonitrile has a lower absorbance in routine HPLC and UHPLC procedures and causes less interference than other solvents that can be used.

Biosolve Solvents in Laboratory Glassware

Biosolve solvents, including Acetonitrile, supplied by Greyhound Chromatography, are filtered at 0.1µm and are packed under inert nitrogen gas for longer shelf life, with very low amounts of inorganic metallic contaminants.  Acetonitrile is bottled in pure glass containers to minimise leaching and degradation of the solvent.  Acetonitrile is a clear colourless liquid with an acidity as low as 0-0.001% depending on the grade.  The presence of acid in the solvent improves the chromatographic peak shape.  Full certificates of analysis are available to view before selecting the right grade of solvent for your application.   Acetonitrile Surpa-gradient grade, available from Biosolve, is the highest-grade Acetonitrile solvent available for HPLC and UHPLC in today's worldwide market. 

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Acetonitrile is shipped under Dangerous Goods regulations as it is a highly flammable liquid and vapour.  Safety procedures should be closely followed for the safe handling of Acetonitrile, including keeping away from sparks and open flames, hot surfaces and heated storage rooms.  When handling take reasonable precautions to ensure that it does not go on skin or near eyes.  If Acetonitrile does come into contact with clothes, remove the clothing and rinse thoroughly in water.  If it comes into contact with the skin, wash cautiously with water.  If acetonitrile comes into contact with the eyes, remove contact lenses if they are present, rinse with water for several minutes and seek medical advice. 

 

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Is there a need to wash columns frequently during HPLC

It is important to look after your columns to prolong their life and also their integrity.  Washing a column is an important process that should be done regularly.  Depending on how many samples you are processing and how often you are using them will determine how many times you wash the column.  Storage of the cleaned columns should also be considered.  It is important to establish a wash cycle in the laboratory, if you do not you will notice a gradual pressure build up and you may experience ghost peaks after the first injection.   Ghost peaks are caused by compounds eluting off the column in later runs despite being injected in a previous analysis. 

This is the recommendation for a column that is to be stored over a long period.  Replace buffers and modifiers at the end of the day, it is recommended that you wash the column with 70% water, 15% methanol and 15% acetonitrile.  divert the eluent from the column to waste so as not to contaminate the detectors.  Wash the column in 100% methanol and 100% acetonitrile, both for at least 15 minutes.  Store the column subject to the manufacturers recommendations, this will preserve the column, which may last for years depending on the number of samples processed and the care you give to your column.   For daily use, wash the column in 70% water, without using an acid or modifier, and store overnight subject to manufacturer's instructions. 

 

Bioslve Vials Image

 

Historic World Shortage of Acetonitrile

In 2008 there was a world shortage of Acetonitrile, (a by-product from the manufacture of acrylonitrile) because of several factors, including, Chinese manufacture being shut down because China was hosting the Olympics and all industry within China was either closed down or drastically reduced in an attempt to improve the standard of air during the Olympics.  Also, during Hurricane Ike in the USA an Acetonitrile factory was damaged in Texas causing further shortages.  As Methanol is an acceptable substitute in most HPLC methods the shortage can be overcome.  Acetonitrile is seen as a valuable commodity due to the volume used worldwide for a variety of purposes other than Laboratory analysis.  There is no anticipated shortage of acetonitrile at this present time.  

 

Author:

Susan Massie, Marketing Director

Greyhound Chromatography and Allied Chemicals

www.greyhoundchrom.com

 

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What is a Ferrule? | Greyhound Chromatography

What is a Ferrule?

Available in materials such as brass, steel, graphite and a variety of different plastics, ferrules connect two components together. The term is primarily tied to scientific experiments and practices, but different versions are also used for holding bristles to brushes, handles to golf clubs, strings to wooden instruments, grips to snooker and pool cues, wooden handles to blades for woodwork tools and rubbers to the ends of pencils. Even the plastic tip at the end of shoelaces is classed as a type of ferrule.

Looking at the more outlandish ways that ferrules have been utilised, electricians and dentists have used them for equipment that require strength, durability and a watertight seal. From a scientific standpoint, ferrules are most commonly used to attach pipes to test tubes, flasks, cylinders and beakers, with gas chromatography ferrules being the most common reason for their use. While they’re only a small component in any scientific process, they are just as important as any other part of your lab equipment, and if you use an unsuitable alternative or choose the wrong ferrule, the results of the scientific process you’re conducting could be made invalid.

What does a ferrule do?

Sometimes simply referred to as rubber stoppers, ferrules are used in laboratories for safely joining two components together, allowing liquids or gasses to pass through tubing without any of the substance escaping. The purpose of a ferrule is to be both airtight and watertight to protect the substance and - if it’s a potentially harmful substance - the people handling it.

You’ll likely recognise rubber ferrules from high school science experiments, where they would have been used to seamlessly support travelling gasses and liquids whilst preventing any spillages for processes including gas chromatography. Not only would this combat mess around the area of your experiment but it would also stop any dangerous substances from coming into contact with the people involved in the experiment.

It would always be advised to use a ferrule for any scientific process that requires a substance to pass between two components, as even a substance that isn’t harmful will need to be secured within the tube or it will be likely to seep out. Ferrules are made from different materials such as metal, rubber and plastic to suit different uses, with the chosen material being based on the specific use. All three of these materials could be used for scientific processes like gas chromatography.

Rubber ferrules and piping in a science laboratory stockroom.

 

What size ferrule do I need?

Identifying the correct size of ferrule you need should be based around the equipment you’re using and the process you’re using it for. Rubber ferrules should usually fit quite tight in the tube you’re connecting them to in order to reliably prevent any substances from escaping and any outside interferences from affecting your results.

All scientific processes must be precise in order for them to return the best results, ruling out any chance of bias or unfairness towards a specific outcome. Even minor components in science equipment like ferrules need to be exact for an impartial result, so there’s an extensive range of different sizes to account for every shape and size of test tube, flask, cylinder and beaker.

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Why Use A Pipette? | Greyhound Chromatography

 Why Use a Pipette?

A common tool within a laboratory environment, pipettes are primarily used for the transportation and measurement of various substances. Also referred to as chemical droppers, while they’re not the first thing that comes to mind when thinking about processes such as gas chromatography, they’re immensely important, offering uses that other similar types of lab equipment do not.

 

Most scientific experiments or processes will make use of plastic pipettes, but it’s also possible to use glass pipettes, with the only difference between the two being a rubber head used at one end of a glass pipette to pull and push a substance in and out of it. With plastic pipettes, on the other hand, you can simply squeeze the larger end of the pipette to do this.

An alternative to both of these traditional types of pipettes are electronic pipettes, where multiple samples can be taken at once. This makes syringing, measuring and transporting chemicals quicker, easier and more accurate.

Why use a pipette instead of a measuring cylinder?

It’s common to use measuring cylinders for handling potentially harmful substances, but there are advantages to using a pipette instead. Unlike a measuring cylinder, a pipette will be more accurate with all of the sample, accounting for every drop of the substance being held within the tool. If there’s a few drops of the sample at the side of the measuring cylinder, these aren’t guaranteed to be included in the total measurement, but a pipette will be as accurate as possible, especially when using an electronic pipette.

 

Health and safety factors when using a pipette are also far less of a concern than they would be when using a measuring cylinder due to how the substances are held. A measuring cylinder is a traditional way of handling, measuring and transporting fluids within a laboratory, but the risk of spillages can be a concern. Choosing to use a pipette means that instead of the substance potentially flowing over the edges of the cylinder, they should remain secure and easily under control inside the pipette, even when being moved around a lab.

An electronic pipette taking multiple samples in a laboratory

How does a pipette measure?

A typical plastic pipette works as a vacuum, with fluid drawn in and out of the tool by squeezing the ‘bulb’. Marked units are clearly labelled along the shaft of this type of pipette, making the process of recording measurements simple. You’ll often find milliliters as the unit of measurement on a plastic or glass pipette - usually ranging from between 0.25 and 1 centimetre - with electronic pipettes going as low as 50 microlitres, which equates to as little as 0.05 milliliters.

 

Electronic pipettes are recognised as being even more accurate than traditional pipettes. The latest electronic pipettes offer a whole range of different settings, meaning that you can set up what you want them to do and they’ll draw in the substance to your exact specifications. Even processes such as dilution and titration are provided as options, and you’re able to choose the exact amount of fluid you want it to draw in if you’re restricted to specific measurements. Additionally, you will be able to control the speed at which you’re drawing a fluid in and out of your pipette, which is ideal if you’re handling a particularly volatile substance.

 

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Tel: +44 (0) 151 649 4000

Web: www.greyhoundchrom.com

Email: info@greyhoundchrom.com

Follow Us

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You may also be interested in 

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How to Prepare a Sample for HPLC Analysis | Greyhound Chromatography

How to Prepare a Sample for HPLC Analysis

 

 

High-performance liquid chromatography (HPLC) is a analytical chemistry technique used to separate, quantify and identify each component in a mixture. Components are mixed in a pressurized liquid solvent which pass the sample through a HPLC column filled with a solid  adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.  The sample is mixed with solvents in varying dilutions and can be pre-filtered before the sample is put into the column to ensure purity of the results, where a sample has to pass through the column at a regular or fast speed a HPLC pump is used.  

 

Sample preparation is vital to high quality reproducable and specific results, often overlooked methodical sample preparation can improve efficiency in the laboratory.  Sometimes seen as a laborious task sample preparation isn't always viewed as the most efficient use of time in the laboratory.  HPLC and increasingly UHPLC is one of the most commonly used high pecision analytical methods in today's fast paced modern laboratory.  A sample needs to be prepared so that it can be directly injected into a HPLC column.  Firstly the sample needs to be dissolved in an appropriate solvent; methanol and acetonitrile are favourite choices, for a variety of reasons; there are many guides available as to the proportion on solvent in relation to your sample; some methods are tried and tested some are just a matter of trial and error.  Chromatography forums provide a wealth of experience that is freely shared via the Internet.

Depending on the sample to be analysed, whether it be for animal biological fluids, drinks, food, human biological fluids, petrochemical residues, pharmaceutical analysis, toxicological residues, waste waters, the sample will need to be filtered prioir to injection.  Solid Phase Extraction (SPE) tubes provide an excellent clean up method or a syringe filter with a suitable membrane to ensure that your sample is free of particles that may cause intereference during detection and will prevent blockages to your column.   Syringe filters are the most efficient method of pre-filtering the sample for sample with complex properties or for small volumes, they come in a variety of membranes and differing porisities.  It is important that the sample is a clean as possible before it is introduced to the column, taking time at this stage to ensure that the sample is a clean as it can be is worth the extra time.  

Problems with peaks, particularly Ghost Peaks can be detected either with the first injection or problems may present themselves during the course of your analysis leading to a gradual deterioration in your results.  A poorly prepared sample may lead to you having to repeat the whole process, it may even involve you having to clean the column if the sample is too contaminated.  Taking extra time to clean the sample will save valuable laboratory time in the long run. 

A clean sample will help to ensure:

  • High sensitivity of the HPLC Column
  • Accurate, reproducable results
  • Fewer false-positive peaks
  • Reduced leachables
  • Low background noise
  • No blockages in the column

Using ultra pure water can also have its advantage particularly for pharmaceutical analysis where the need for the purest water is paramount.  Scientific research has proved that more than 80% of problems experienced during HPLC analysis are directly attributable to water purity.  Biosolve Ultra Pure water is supplied by Grehoyhound Chromatography in the UK and to laboratories world-wide.  With less than 0.0002%w/w residues after evaporation, the HPLC grade water from Biosolve is filtered through 0.2µm and bottled under inert gas.  

 

Some scientists prefer to use an automated sample preparation methods to eliminate human error, samples can become unreliable where dilution has been carried out manually use hand to eye determination of sample size and not a regulated reproduction of a sample using a elctronic pipette, for example.  Automated sample preparation can aid productivity by speeding up the analysis process, this is partciularly useful for UHPLC where speed of analysis under high pressure is essential.  Triple quad systems provide increased sensitivity at lower analyte concentrations, allowing high throughput of smaples.  The laboratory environment is increasingly complex, particularly in the field of biological research and analysis.  The need for research and drug development in the area of Cancer research alone is driving new developments every day at the edge of treatment devlopment.