Getting Started
From TheLiquidPhase
Getting started with CCC can be somewhat intimidating at first. The following quote delivered by A.J.P. Martin more than a half-century ago is as true now as it was then:
"I would like to take this opportunity to record here my belief that one should take a minimum of care and preparation over first experiments. If they are unsuccessful one is not then discouraged since many possible reasons for failure can be thought of, and improvements can be made. Much can often be learned by the repetition under different conditions, even if the desired result is not obtained. If every conceivable precaution is taken at first, one is often too discouraged to proceed at all."
-Archer J. P. Martin, December 1952 (Nobel Prize acceptance speech, awarded for his work on the development of partition chromatography)
Most modern CCC instruments are quite robust. It would be quite difficult for a user who has basic common sense to do any permanent damage. It is highly recommended that the new user gain familiarity with running the instrument, monitoring system pressure, and measuring stationary phase retention prior to injecting a precious sample.
Contents |
Learn
The first step of your journey into the world of CCC should be to learn everything you can. This site, of course, is an excellent place to start, and links out to nearly every other resource on the internet. Also, you will find it helpful to review some of the many Publications in scientific journals that relate to CCC.
Develop a Support Base
Begin to develop a network of colleagues and/or service providers that will be there when you need help.
Choose an instrument Type
Choose an instrument
Speed, column volume, flow rate, sample loading, etc., are all things that should be considered when choosing an instrument. However, it is important to know how these numbers fit together and are used to determine total machine performance and characteristics. There are relationships between these properties that, together, become very important. For example, throughput, one of the most important characteristics for commercial organizations, can be calculated if you know: (1) How much you can inject, (2) how fast you can pump mobile phase through the system (while retaining sufficient stationary phase), and (3) the partition coefficient of the target compound.
Here is a list of some of the most important characteristics to consider when buying a CCC:
Throughput
Throughput is the one variable that most closely corresponds with instrument cost. It directly relates to the human hours required to operate the machine. Further, it is important to be aware of the broad range of throughput currently available.
Lets take for example the Pharma-Tech CCC-1000, with the largest (850ml) coil. According to their specs, the best case with this machine will allow you to load about 2g per injection, with a flow rate of 5ml/min. This translates to 340 minutes (more than 5 1/2 hours) to elute to a partition coefficient of 2 (a typical run). If you include the time required to mix the solvents, fill and equilibrate the machine, and deal with isolated fractions, it takes one entire workday for one run on this machine. To put this in perspective, other machines can accomplish the same separation with >10x the injection weight, in less than an hour. Further, our industrial scale machines can actually do significantly more than that.
The bottom line... Determining the right machine will depend on how much material you need to purify and how you or your entity values time vs. money. Also, keep in mind that your actual throughput will depend on your specific application. Different sample matrices will have different effects on the stationary phase retention, which will vary your injection capacity, as well as the rate at which you can pump mobile phase. Work with your vendor to determine what machine might best work for you and your application's needs.
Resolution
Sample resolution depends on a number of factors: the properties of the sample and phase systems, the number of mixing and settling cycles per unit time, the rate of mass transfer during mixing, and the quality of mixing between the phase systems. Resolution to a large degree corresponds to the effective g field produced by the centrifuge, which in turn is related to the cost of the instruments.
Manufacturing Quality
The newest CCC machines do not have the same issues of poor reliability and maintenance as do generations past. They can now easily go through months of heavy use with little to no maintenance, very unlike past machines that require maintenance at least weekly. Modern machines also provide technology advances like temperature controlled separation chambers. The advent of which has grown to be an important issue in repeatable separations.
Here are some helpful questions to ask when considering an instrument:
- How often is regular maintenance required?
- Flying leads?
- Bearings?
- How easy are common maintenance routines?
- Is a visit from a service provider recommended and at what frequency?
- What are the most common problems with the machine?
- Can I get replacement parts if needed?
- Are there technicians available to do difficult part replacements?
Scalability
Another question you need to ask yourself is if you or your company needs the option of fast scale up to production levels. With modification to the original method, most separations can be recreated on any CCC. However, certain manufacturers maintain system variables within tight tolerances, which allow the successful use of essentially the same method across their entire product line. If you are likely to scale your projects up to production levels, consider a manufacturer that tailors to such.
Safety
Safety is always an issue in the laboratory. And, like any other instrument, it is important that users understand how the instrument works in order to avoid damage to the machine, or worse, the user. Similar to cars that have anti-lock breaks and air bags, modern CCC systems can provide many safety features, such as:
- Strong and robust construction materials, which would contain any mechanical failure.
- Automatically locking doors, which prevent human contact with moving parts.
- Automatic leak shutdown.
- Automatic out of balance shutdown.
- Automatic temperature shutdown.
Availability of these systems vary from machine to machine, but it is important to know that they exist. Further, as stated above, it is very important that the user know how to properly operate the machine. In this regard, you want to make sure that you will be provided with sufficient on-site training.
Support Availibility
It is important to note that, because CCC has only recently become a highly-viable technique, the availability of qualified support/knowledge is limited. You will probably want to investigate your vendor's ability to provide fast, responsive and knowledgeable service. Further, do they provide a convenient way for you to contact them during your work hours?
Peripheral Equiptment
Like any other LC system, CCC instruments require peripheral equipment (See A Typical Setup). Solvent pump, detector, fraction collector, chiller, etc.. If it is important to you or your company, you may want to find out if the vendor provides this equipment, or if you will need to deal with outside vendors. Further, if your vendor does not carry this support equipment, will they, and do they have the ability to, make recommendations?
Warranty
Most CCC instruments are backed by at least a 1 year manufacturer, parts and labor warranty; and some offer lifetime warranties on certain key parts of the system. However, this is something you should definitely check out.
Selecting and Purchasing Solvents
One of the most commonly used solvent systems is the HEMWat family (1,2). In order to take advantage of the voluminous literature describing sucessful separations of virtually every compound class, it is highly recommended to have stocked hexane (or heptane or petroleum ether), ethyl acetate, methanol, and a source of purified, deionized water.
Moving On
Once you have an instrument you are ready to move on to Your First Run.