SubscribeAmphotech Ltd. has numerous offerings for HPLC in Bioscience, collectively we have over 30 years experience in complex HPLC separations applications and electromechanical HPLC instrumentation expertise.
The Services & Instrumentation & Equipment that we offer includes:
Contract Research & Fee for service HPLC separations for analysis & purifications
HPLC Methods Development
Technical training for operation & maintenance of HPLC’s, Methods development, & HPLC applications technical consulting services.
With specific manufacturer affiliates we sell brand new latest versions of their licensedHPLC Software & Interfaces for data acquisition and control of Waters, Hewlett-Packard, Rainin, Varian systems and soon Shimadzu, and others.
HPLC System Refurbishment & Upgrade services & locally offer on-site services. Pre-owned HPLC Equipment instrumentation including entire systems and component units, we buy and sell HPLC systems, and on limited basis provide set-up installation & training services.
Soon Amphotech Ltd. Will offer brand new HPLC columns & accessories to our customers, our choices of any new products we sell are always based upon technical comparisons of quality and performance.
Amphotech Ltd. also provides referral services for HPLC mobile phase solvents & reagents, HPLC consumable OEM parts and we assist our clients with equipment components searches.
Overview of High Pressure Liquid Chromatography (HPLC) Technology & Separations
In today’s science there is an ever growing need to visualize and characterize am ever increasing number of substances as we investigate our environment and isolate purify or create molecules and substances by numerous methods.
HPLC technology has greatly evolved to offer numerous methods and analytical capabilities. Numerous types of detectors attest and include:
Ultra Violet UV
UV-VIS
Fluorescent
Refractive Index (RI)
Chemical Ion
Evaporative Light Scattering (ELSD)
LC-MS ( HPLC-Mass Spectrometry )
A discussion of these methods of detection follows comparing their aspects:
Ultra-Violet & UV~VIS Detection:
UV detection is achieved via an optical fluid flow cell in the pathway of a UV light source and a UV detector cell that through the detector module measures the U.V. Absorbance’s
of substances passing through the flow cell and outputs an analog signal in millivolts that are converted to milliabsorbance units (mau’s) by either an integrator or a data acquisition software & computer system. Integrators produce a chromatogram plot plus mark peak elution time and calculate peak area or peak height depending on program choices.
U.V. and UV~VIS detectors are the most commonly utilized types in HPLC work, and the majority of scientific papers discussing separations and purifications are based upon using this type of detection. There is as a result a vast array of knowledge about a wide variety of compounds and substances and their separations characteristics.
Numerous databases have been created by researchers, column manufacturers, and there are data bases of chromatograms through U.S. NIST of standards and highly pure substances.
UV~VIS detectors utilize either dual lamp sources or a single wide range lamp type to provide Ultra-violet and visible range measurements. For example in protein and peptide work 210nm and 280nm UV wavelengths are utilized to look at intra-protein, intra-peptide amino acid bonds and aromatic amino acids respectively. While the visible wavelength can be use to observe at 570nm Ninhydrin (Kaiser Reaction) produced derivatized alpha and epsilon amino termini of the n-terminus of a peptide or protein or the epsilon amino termini of Lysine's
The millivolt output from the detectors can also feed to a strip chart recorder including single or dual channel units to produce a simple plot of the chromatograph on strip chart graph paper. The type of graph paper and strip chart speed and sensitivity settings are utilized to observe elution time and peak characteristics and may allow manual calculation of peak area or peak height in millivolts to compare to the results of running standards of known purity and concentration
Some U.V. detectors offer dual wavelengths and are utilized in protein and peptide chemistry HPLC analysis. For example intra-peptide or protein amino acid bonds are typically measured at 210nm, while aromatics such as found in Tryptophan and Tyrosine are measured at 280nm. The dual wavelength detector measures both of these wavelength absorbance’s continuously throughout a separations run.
In protein chemistry work enzymatic or chemical digests such as Typsin, Chymotrypsin and Cyanogen bromide respectively produce a mixture of peptide fragments that are separated typically on a solid phase of C18 4.5mm x 150mm 3.0 micron particle size column. The typical gradient utilized is 0.05% TFA aqueous to 0.06% TFA in either 80% MeCN: Aqueous or 100% MeCN mobile phase.
Fluorescent Detectors:
These work on the same principles as U.V. Detectors but have special programs allowing excitation wavelength to pass through the flow cell while utilizing more sensitive detection needed for precise work on fluorescently labeled compounds. Dual detection systems allow measurements of indirect and direct fluorescence on certain instruments via these and optimization of light pathways.
Refractive Index Detectors:
The refractive index of a substance is the degree of light refraction that it produces, by measuring this under precise conditions the Refractive Index can be compared to those obtained with standards. In clinical medicine “Refractometers” are utilized to measure specific gravity S.G. on body fluids and the standard to which these are compared is distilled deionized water that has a specific gravity of 1.000. So in essence the amount of dissolved substances and thus, Specific gravity has been correlated to the refractive index and the results are used to diagnostically evaluate states of hydration and organ function such as Kidney renal function aspects (ability to concentrate via dialysis processes). Of course the measurement of dissolved substances specific gravity can be observed in many different types of materials utilizing refractive Index Detectors coupled with HPLC separation to produce analytical characterization.
Chemical Ion Detectors:
These detectors utilize ion selective electrodes that are implanted into the flow cell to measure specific ions from substances that elute from the HPLC column. This measurement is greatly influenced by presence of various metal ions and requires either Teflon HPLC pump heads and use of “Peak” plastic tubing’s and or special in-line
metal trapping filters as the metal ions will interfere with measurement of target ions via quenching of signals or via poor signal to noise ratios and suboptimal detection.
Evaporative Light Scattering Detectors (ELSD):
Commonly described as a poor man’s Mass Spectrometer this unique type of detector offers amazing capabilities to measure a wide variety of substances. Many substance types do not produce any U.V.-VIS absorbance’s and cannot be observed on typical UV based HPLC detection systems. The ELSD detector is basically quite similar to an electrospray part of an electrospray ESI Mass Spectrometer. In that there is a heated chamber and a flow of a carrier dispersing gas such as high grade Argon or ultra pure Nitrogen gas that served to atomize the HPLC eluant liquid mobile phase flow upon arrival and the atomized spray is then passed through a detector with a laser energy pulse that excites the molecules which in turn scatters the light. The light scattering is measured by a high sensitivity or series of detectors and compared to a reference beam measurement.
Unlike ESI Mass Spectrometers however, there is absence of a high vacuum system and time of flight tube that facilitate mass measurement. There are companies that have developed instruments that can convert ELSD data to calculate molecular mass. Yet the mass accuracy is reported as good for rough estimations of molecular weights, there are many variables and the mass accuracy is significantly lower than that achievable via true mass spectrometry. For example such a system may assign a mass of 56Kd to a protein but the actual mass of the protein may be 52Kd so it certainly good for screening and basic evaluations of HPLC peak isolates.
The advantages of ELSD are that it allows visualization of certain types of molecules that for example do not have UV Absorbance’s these include types of Glycosides. Further advantage is that it can provide ballpark mass determinations of peak isolates by comparison to results obtained running known molecular weight standards producing more accurate results than Electrophoresis running molecular weight standards.
LC-MS (HPLC-MassSpectrometry)
The most common type of LC-MS is via conversion of an electrospray system to allow sample introduction via microflow HPLC to the electrospray atomization chamber versus direct sample introduction via a syringe pump system. LC-MS allows that an optimized HPLC separation be effected to separate different components allowing their discrete analysis by mass ionization. LC-MS systems typically utilize either a photodiode array UV-Scanning detector or a UV detector between the column outflow and the atomizer chamber. Thus, both the absorbance trace and the mass spectra data are observed and recorded. Through various software most often from system manufacturers the HPLC chromatogram peak isolates shown from the HPLC detector can be mass labeled as well as labeled with the elution time, and the text report provides peak area.
In the case of systems with a photodiode array scanning UV-VIS detector the absorbance’s spectra for all significant wavelength absorbance’s (per programming choices of milliaborbances minimum and maxima) is plotted either via color plotting using different colors or via solid, dashed or dotted etc. lines in the case of monochrome. Thus, the absorbance maxima for unknown UV-VIS light absorbing compounds, the elution time, the peak area’s at various wavelengths and the mass of the eluted peak are all determined and present in the data. Examination of both peak symmetry and various absorbances’ determine the relative purity of eluted compounds. Peak area compared to that of standards allows analytical quantitation and mass analysis determines the molecular weight of each eluted compound isolate.
HPLC Separations Technology:
The separation of components, molecules, substances and analytes is the goal of a chromatographic separation process. This goal is achieved by careful analytical work. In the choice of and the preparation of the mobile phase liquid solvent buffers, choice of the type of solid phase column, and choice of the gradient conditions between buffer solvents. The choice of flow rate, depth of gradient, slope of gradient, column particle size and hydrophobic interaction solid phase type are combined with sample measurement and introduction choices, column heater temperature control, and the type of detector and its settings and type of data acquisition utilized to produce analytical results.
Photo of Refurbished Waters HPLC 600 Pump & Controller Module
Analytical results can then be utilized for characterization interpretations or for scale up preparative purification work on larger quantities of any given material as long as the material is soluble in compatible liquid solvents.
Methods Development:
In considering performing HPLC on any type or class of materials the first aspect is a thorough examination of scientific literature about these materials and specifically evaluating published accounts of their separations characteristics. In many cases various methods may have been utilized and comparing the actual chromatograms will provide great insight towards choosing the best method.
In many cases the scientific papers found and data and results examined will suggest more than one way to run HPLC on a specific type of material. It is then that the chromatographer would perform comparison runs and evaluate chromatograms produced.
For unknown or novel compounds it may require that numerous runs be made with different mobile phase solvents, different gradient types, different column types, and even different detector types.
The literature search for the type of most similar compounds results will act as a guide to narrow down the number of different methods to run and compare.
Method Optimization:
The separation can be optimized by a complete methods development with particular attention to adjusting the gradient slope and time making gradients more shallow or steep will effectively zoom-in on the region of buffer solvent mobile phase concentrations and allow the degree of gradient change (% organic solvent concentration) to change more slowly effecting better separation of individual constituents.
It is my hope that you enjoyed this short overview of this exciting technology... If you have questions or would like information about services or instrumentation and equipment please contact me.
Sincerely,
Neal H. Wright
President & CEO
R&D Lab Director
Amphotech Ltd.
100 Cummings Center
Suite 426H
Beverly, MA 01915