Monday, April 12, 2010

PCR











PCR TYPES
PCR Troubleshooting
PCR Problems and Solutions, PCR Help
What is your PCR problem?:
Long Non-specific PCR products?
Primer Dimer formation?
Problem:
Long Non-Specific Products in PCR
When running an agarose gel you spot larger non-specific PCR bands that are not the right size.
Solutions to Long Non-specific Products in PCR:
Decrease annealing time
Decrease extension time
Decrease extension temperature to 62-68º C
Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM
Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.
Use less primer
Take less DNA template
Take less Taq polymerase
If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)
Combination of some/all of the above.

Problem:
PCR Primer Dimers
When running an agarose gel, you spot some very small PCR bands. These are the size of your primer or about the size of both your primers (A and B) together. These are termed "primer dimers" and are formed by the annealing of your primer with itself or with the other primer.
Solutions to primer dimers in PCR:
Use less primer.
Re-design primer and order a new batch. Make sure you use primer design software and check for self-annealing and that the primers do not share a large percentage of complementary sequence. Check primers carefully for homo-dimer and hetero-dimer formation with OligoAnalyzer
Conduct PCR with and without formamide.
Titrate Mg2+ (MgCl - 1.5, 2.0, 2.5 and 3.0 mM) concentration.
Increase DNA template amount (concentration).
Increase annealing temperature (try optimizing using a gradient PCR machine to find optimal temperature for annealing).
Try adding DMSO up to 5%.
Try using HotStart PCR instead of regular Taq polymerase PCR.
Combination of some/all of the above.
AFLP PCR Background
Amplified Fragment Length Polymorphism PCR, also called AFLP PCR was originally described by Zabeau et al., 1993.
AFLP is composed of 3 steps:

1-A) Cellular DNA is digested with one or more restriction enzymes. Typically this involves a combination of two restriction enzymes: a 4 base cutter (MseI) and a 6 base cutter (EcoRI).
1-B) Ligation of linkers (restriction half-site specific adaptors) to all restriction fragments.
2-A) Pre-selective PCR is performed using primers which match the linkers and restriction site specific sequences.
2-B)
3) Electrophoretic separation and amplicons on a gel matrix, followed by visualisation of the band pattern. The aim of this tool is to perform a theoretical AFLP-PCR experiment by using the same principles, and to suggest the adaptors and primers needed in the experiment.
Applications of AFLP PCR
AFLP is a highly sensitive PCR-based method for detecting polymorphisms in DNA. AFLP can be also used for genotyping individuals for a large number of loci using a minimal number of PCR reactions.
Allele-Specific PCR Protocol
Allele-specific PCR is a varation of the polymerase chain reaction which is used as a diagnostic or cloning technique, to identify or utilize single-nucleotide polymorphisms (SNPs) (single base differences in DNA). Allele-specific PCR does require the sequence of the target DNA sequence, including differences between the alleles.
Primers for Allele-Specific PCR
The allele-specific PCR uses primers whose 3' ends encompass the SNP.
PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with a SNP-specific primer signals presence of the specific SNP in a sequence.
Assembly PCR
Assembly PCR is a polymerase chain reaction variation that artificial synthesizes long DNA sequences by performing PCR on a pool of long oligonucleotides (primers) with short overlapping segments.
The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final long DNA product.
Asymmetric PCR
Asymmetric PCR is used to preferentially amplify one strand of the target DNA more than the other.
Applications of Asymmetric PCR
The Asymmetric PCR is useful in some sequencing and hybridization probing applications where having only one of the two complementary stands is sufficient or required.
Asymmetric PCR Method
The asymmetric PCR method is conducted as the standard pcr protocol, however a great excess of the primers for the chosen strand is used.
Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.
Also see Linear After the Exponential PCR or LATE-PCR.
Primers for Asymmetric PCR
A PCR in which the predominant product is a single-stranded DNA, as a result of unequal primer concentrations.
As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into double-stranded DNA. The higher concentration primer continues to primer synthesis, but only of its strand.Troubleshooting Asymmetric PCR
Helicase-dependent PCR
Helicase-dependent amplification (HDA) is a method which is different but is based on the PCR.HDA uses a constant temperature instead than cycling temperatures through denaturation and annealing/extension cycles. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.
Applications of Helicase-dependent amplification
Helicase-dependent amplification.Troubleshooting HDA Helicase-dependent amplification
Inverse PCR
Inverse PCR Background
Inverse PCR also called IPCR, and was first described by Ochman et al. in 1988 (1).
A limitation of standard PCR is that 5' and 3' flanking regions of your DNA fragment of interest must be known. Inverse PCR allows you to conduct PCR when you only have the information of one internal sequence.Inverse polymerase chain reaction is a variant of PCR, and is used when only one internal sequence of the target DNA is known. It is therefore very useful in identifying flanking DNA sequences of genomic inserts. Similar to other PCR methods, inverse PCR amplifies target DNA using DNA polymerase.

Inverse PCR uses standard PCR (polymerase chain reaction), however it has the primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle.
The Inverse PCR Method
The inverse PCR method includes a series of digestions and self-ligations with the DNA being cut by a restriction endonuclease. This cut results in a known sequence at either end of unknown sequences.
Inverse PCR Steps
1) Target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion.2) Self-ligation is induced under low concentrations causing the phosphate backbone to reform. This gives a circular DNA ligation product.3) Target DNA is then restriction digested with a known endonuclease. This generates a cut within the known internal sequence generating a linear product with known terminal sequences. This can now be used for PCR (polymerase chain reaction).4) Standard PCR is conducted with primers complementary to the now known internal sequences.
In summary:
Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested and then ligated to generate circular DNA.
PCR primers pointing away from the known sequences are then employed to amplify the flanking sequences.
Applications of Inverse PCR
Inverse PCR has numerous applications in molecular biology including the amplification and identification of sequences flanking transposable elements, and the identification of genomic inserts.
In Situ PCR
Definition of In Situ PCR
In Situ PCR (ISH) is a polymerase chain reaction that actually takes place inside the cell on a slide. In situ PCR amplification can be performed on fixed tissue or cells.

In Situ PCR Introduction
During the initiation and progression of disease, minute quantities of a product in small populations of cells or tissues may be vital for the pathogenesis of the disease.
In many slowly-evolving diseases which require months or even years to manifest themselves clinically, it has been shown that the majority of the affected cell population is in a transcriptionally inactive state, and at a level of one gene per host cell.
Nucleic acid hybridization methods and the polymerase chain reaction (PCR) have both been employed to examine the expression and detection of such affected genes during pathogenesis. While both these techniques are quite useful, the disadvantage of these techniques is that they are essentially conducting cell expression and population studies. Nucleic acids are isolated from a population of cells which contains either a sufficient number of molecules to detect directly by standard hybridization techniques, or, when a subpopulation contains as little as a single copy of nucleic acid, that molecule amplified by the PCR, and detected after amplification.
In situ hybridization (ISH) applies the methodology of the nucleic acid hybridization technique to the cellular level. Combining cytochemistry and immunocytochemistry, In Situ PCR allows the identification of cellular markers to be identified, and further permits the localization of to cell specific sequences within cell populations, such as tissues and blood samples.
In Situ PCR is limited to the detection of non-genomic material such as RNA, genes or genomes, as the detection limit in most conditions is several copies of the target nucleic acid per cell. Therefore, due to copy number limitations, hybridization of RNA is more sensitive than DNA detection.
Factors affecting In Situ PCR sensitivity include:
1) the strandedness of the target molecule
2) the lack of a complementary sequence proximal to the target sequences
Reverse transcriptase-catalyzed in situ transcription has even been used to detect RNAs which occur at relatively high copy number.
Intersequence-specific PCR or ISSR
Intersequence-specific PCR or ISSR Protocol
Intersequence-specific PCR or ISSR PCR is a method based on the polymerase chain reaction.
Applications of ISSR PCR
The ISSR PCR method is used for DNA fingerprinting by amplifying regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths. Troubleshooting Intersequence-specific PCR or ISSR
Late-PCR
Linear-After-The-Exponential-PCR Protocol
A recent modification of assymetric PCR is Linear-After-The-Exponential-PCR or LATE-PCR.
Applications of Asymmetric PCR
The Asymmetric PCR is useful in some sequencing and hybridization probing applications where having only one of the two complementary stands is sufficient or required.
Asymmetric PCR Method
The asymmetric PCR method is conducted as the standard pcr protocol, however a great excess of the primers for the chosen strand is used.
Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.
Also see Assymetric PCR.
Primers for LATE-PCR
LATE-PCR utilizes a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.[16]
Troubleshooting LATE-PCR
Late , long pcr types nested pcr
Real-Time PCR
REAL TIME PCR : An Introduction
Although the traditional methods of quantitating mRNA are fairly good such as northern blotting and in situ hybridization, they do not approach the ease and speed of Real Time PCR.
RT-PCR or reverse transcriptase PCR is semi-quantitative due to need to load samples on a gel and the insensitivity of ethidium bromide. Thus, real time PCR was developed out of the need to quantitate differences in mRNA expression in a easy and quick manner, and due to the need to use of small amounts of mRNA such as those obtained by small tissue samples, and LCM (laser capture microdissection) isolated cells.
Real-time reverse-transcriptase (RT) PCR is different from other quantitative PCR as it quantitates the initial amount of the template instead of detecting the amount of final amplified product (Freeman, 1999; Raeymaekers, 2000).
Real Time PCR is characterized by the point in time during cycling when amplification of the PCR product of interest is first detected rather than the amount of the PCR product of interest which is accumulated at the end-point after PCR which contained a large number of cycles. Real Time PCR does this by monitoring the amount of fluorescence emitted during the PCR reaction, and this acts as an indicator of the amount of PCR amplification that occurs during each PCR cycle. Thus, in newer Real Time PCR machines, one can visually see the progress of the reaction in "real time".
Real Time PCR also has a much wider dynamic range of up to 107-fold (compared to 1000-fold in conventional RT-PCR). The dynamic range of an assay determines how much the target concentration can vary and yet still be quantified. This wide dynamic range also results in a more accurate quantitation.
RT-PCR
Reverse Transcription Polymerase Chain Reaction
Reverse transcription polymerase chain reaction (RT-PCR) is based on the polymerase chain reaction (PCR). More importantly it is based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses.
The techniques of RT-PCR allows the formation of cDNA (complementary or copy DNA) from RNA, which stores the sequence of RNA (such as messenger RNA, mRNA) in the more stable form of nucleic acid, DNA. This reverse transcription from RNA into its reverse complement DNA (cDNA) is the first step of a usually two-step process of RT-PCR. Furthermore, by copying the RNA into DNA, one can then amplify the cDNA sequence by using primers specific for the DNA sequence. This amplification is the final second major step of the two-step process of RT-PCR.
The Process of RT-PCR
The First step of RT-PCR is referred to as the "first strand reaction". In the first-strand reaction, complementary DNA also termed cDNA, is made from the messenger RNA template of interest using oligo dT (oligonucleotide poly-dTs act similar to primers and bind to the 3' polyA sequence located at the 3' UTR - untranslated region, which are present in most mRNAs), dNTPs, and an RNA-dependent DNA polymerase, reverse transcriptase, through the process of reverse transcription.
These factors are combined in a reverse transcriptase buffer for 1 hour at 37°C. After reverse transcriptase reaction is complete, and the cDNA has been synthesized, RNaseH is added (an RNA digestion enzyme) which digests the RNA away from the RNA-cDNA hybrid. After incubation with RNaseH, standard PCR or polymerase chain reaction is conducted using DNA oligo primers specific for the sequence of interest. This second step is referred to as the "second strand reaction".
Thus by adding the thermostable DNA polymerase, upstream and downstream DNA primers, the single stranded DNA becomes double stranded and is amplified, allowing the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA.
Applications of RT-PCR
The exponential amplification of complementary sequence of mRNA or RNA sequences via reverse transcription polymerase chain reaction allow for a high sensitivity detection technique, where low copy number or less abundant RNA molecules can be detected. It is also used to clone mRNA sequences in the form of complementary DNA, allowing libraries of cDNA (cDNA libraries) to be created which contain all the mRNA sequences of genes expressed in a cell. Furthermore, it allows the creation of cDNA constructs which were cloned by RT-PCR and allow the expression of genes at the RNA and protein levels for further study.
Single Cell PCR
Applications of single cell PCR
The invention of the polymerase chain reaction (PCR) has altered the way molecular biology can be studied. With PCR, it is now possible to amplify and examine minute quantities of rare genetic material: the limit of this exploration being the single cell.
Single cell PCR has applications in many areas, and has great application especially in the field of prenatal diagnostics. In prenatal diagnosis, single cell PCR has made possible preimplantation genetic analysis and the use of fetal cells enriched from the blood of pregnant women for the assessment of single-gene Mendelian disorders. Single-cell PCR has not only proven its usefulness in diagnostics, but also lately has been very useful to basic scientists investigating immunological, neurological and developmental problems.
Recent advances in molecular biology techniques such as whole genome amplification (WGA) procedures and single-cell complementary DNA arrays will permit the genetic analysis of single cells to become a more common practice, thus creating new avenues for diagnosis and research.


Single Cell PCR kits
Sigma WGA whole genome single cell PCR kit GenomePlex®
Single-Tube PCR Kit from Takara
The Alliance® HPLC
The Alliance® HPLC System is the flexible and reliable workhorse that meets your fundamental HPLC separation requirements. The system has been continually expanded and perfected to accommodate an enormous range of real-world analytical challenges.
The Alliance HPLC System meets the rigorous requirements of timely and predictable routine analyses in addition to the performance standards of new-product research and development. Alliance systems and columns are manufactured to a rigid set of performance specifications, allowing you to confidently transfer a method between instruments and still get consistent results, unit-to-unit and lab-to-lab.
Whether your lab analyzes samples in the pharmaceutical, chemical, food safety, environmental, or quality control arenas, the Alliance HPLC system offers you a field-proven solution. It offers:
· Integrated solvent and sample management to ensure consistent system-to-system performance and high reproducibility
· Full integration with either Empower™ or MassLynx™ Software for instrument control and data processing
· A large, intuitive LCD-based user interface allows rapid system set-up through SystemPREP routines that streamline daily start-ups
· Easy, tool-free maintenance resulting in maximized uptime
· Waters complete line of detection technologies – from routine UV/Vis detectors to single, tandem and triple quadrupole mass spectrometers - extend your application capabilities

e2695 Separations Module Breeze hplc
The heart of the Alliance® HPLC System is the e2695 Separations Module and its integrated solvent and sample management functions, which ensure consistent system-to-system performance and high reproducibility.
Solvent Management
The module’s solvent management system degasses and blends up to four chromatographic solvents in precise proportions for smooth solvent delivery without pulsing. It offers:
· Serial flow path with only two inlet check valves reduces complexity and minimizes downtime
· Vacuum degassing uses efficient second-generation polymer membrane technology
· Precise low-pressure quaternary blending (first in-first out) provides reproducible gradient profiles across the flow range
· Independently driven pistons produce pulse-free solvent flow without use of pulse dampeners
· Fixed delay volume (regardless of system backpressure) for consistent, predictable chromatography
· Programmable flow rate range covers two orders of magnitude (from 50 µL to 5 mL per minute) without hardware modifications
· Tool-free routine maintenance on plungers, seals, and seal-wash
· Automated active plunger seal wash
Sample Management
Up to 120 industry-standard vials can be accommodated in the e2695’s five sample carousels. Sample queues are quick to set up—whether it’s just one sample or a sequence of multiple methods from different analysts.
· Injections are reproducible from one to hundreds of microliters—compatible with any analytical scale LC column chemistry
· Variable volume injections eliminate the need to swap loops for analytical methods
· Carryover is managed by a programmable needle wash cycle and an active needle wash solution (without wash vials)
· Programmable inject-needle height accommodates various vial geometries and takes into account vial bottom thickness
· Programmable syringe draw rate for viscous samples/solvents
· Five independent carousels allow you to run samples in one carousel while prepping the next sequence in another, enabling new samples/carousels to be added into the system—without disturbing the ongoing sample queue
· Sampling routines for automated reference peak addition or automated pre-column derivitization procedures
· Tool-free lower seal-wash frit and injection syringe replacement, for easy routine maintenance
The Breeze™ 2 HPLC
The Breeze™ 2 HPLC System delivers technology and performance in an affordable, compact, and user-friendly system platform. Complete with software, pump, detector and injector, the system comes pre-configured for different levels of HPLC operational needs.
From teaching purposes to daily analytical work, the Breeze 2 HPLC System integrates simplicity, sensitivity, accuracy, and reliability. It is ideal for any organization seeking a quality HPLC platform with limited budget and chromatography experience including university laboratories, government laboratories, or start-up companies.
Features:
· A completely flexible system that is customizable for any laboratory with quality HPLC components options: binary or isocratic pump, automated or manual injector, UV/Vis, refractive index, multi-λ fluorescence, evaporative light scattering, or photodiode array detector
· Intuitive interface with Breeze 2 Software that allows for easy set-up, operation, and minimal training
Breeze 2 Systems can be configured to fit your application and make it easy to choose the ideal pump, injector and detectors for your needs.
Breeze 2 Software provides access to High Performance Liquid Chromatography with high performance ease. Four simple screens provide real-time monitoring and maintain total control of your system.
Using a single window, Breeze 2 Software provides simplified tools for:
· Control of your chromatographic instrumentation
· Acquisition of chromatographic data
· Interaction with and processing of your data
· Comprehensive reporting
Breeze 2 Software wizards walk you through the steps of creating and managing user accounts, chromatographic systems, and projects for storing your chromatographic data.
Columns and Consumables
ANALYTICAL Columns
Waters is on the forefront of modern HPLC & UPLC® column technology. Our industry-leading columns eliminate significant time and cost per sample from your analytical process—while improving the quality of laboratory results.
As one of the world’s few primary column manufacturers, our ability to control column to column reproducibility, as well as produce some of the most innovative new chemistries, is second to none in the industry.
Particle Technology
In 1993, Waters made a conscious decision to invest in particle synthesis technology and moved to becoming a primary manufacturer of chromatographic particles. The ability to synthesis our own materials brought several significant advantages:
· Complete traceability from final product to raw materials
· Vastly improved reproducibility of final product batch-to-batch and year-to-year
· Flexibility to develop and introduce innovative particle technologies to support an increasing demand for new separation solutions.
Highlighted below are the latest innovative particle technology solutions from Waters.


ACQUITY UPLC Particle Technology
There is more to creating a UPLC® particle than synthesizing a small particle. Many HPLC particles do not possess the mechanical stability and structural integrity to withstand UPLC operating pressures (e.g., 15000 psi/1000 bar). Why is pressure tolerance important? In order to realize the efficiency gains of sub-2 µm particles, the ability to routinely operate at higher linear velocities (e.g., higher flow rates) is required. These higher linear velocities combined with small, sub-2 µm particles result in higher operating backpressures. Waters has created two highly efficient, pressure-tolerant UPLC particles:
· 1.7 µm Ethylene Bridged Hybrid (BEH) particle
· 1.8 µm High Strength Silica (HSS) particle.
The first ACQUITY UPLC® particle created was the 1.7 µm Ethylene Bridged Hybrid (BEH) particle. This second generation hybrid particle is one of the key enablers behind UPLC technology and is available in five column chemistries:
· C18
· C8
· Shield RP18
· Phenyl
· HILIC
Because this is a hybrid particle, a wider usable pH range (up to pH 1-12) makes method development faster and easier. BEH particles are also available in HPLC particle sizes (2.5, 3.5, 5, and 10 µm) in the XBridge™ family of HPLC columns, thus allowing seamless transfer between HPLC and UPLC separations.
The second and newest UPLC particle from Waters is the 1.8 µm High Strength Silica (HSS) particle. As more separation scientists around the world realize the benefits of UPLC technology in their applications, Waters continues to provide additional UPLC particle and chemistry solutions to meet these demands. Waters material scientists developed a High Strength Silica (HSS) particle with the high mechanical stability and appropriate morphology necessary to provide long column lifetimes and UPLC efficiencies at pressures up to 15000 psi (1000 bar). This 1.8 µm UPLC HSS particle is designed and tested specifically for use in UPLC separations and is available in 3 column chemistries:
C18
C18 SB
T3
ACQUITY UPLC HSS C18 chemistry is an ultra-performance general purpose C18 bonded phase that provides superior peak shape for bases, increased retention (vs. ACQUITY UPLC BEH C18 columns), and extremely long column lifetimes at low pH.
ACQUITY UPLC HSS C18 SB columns combine an unendcapped intermediate ligand density C18 bonded phase with the rugged and efficient HSS particle to produce UPLC columns that provide alternate selecitivities for basic compounds under low pH conditions.
An aqueous mobile phase compatible C18 bonded phase designed to retain and separate polar organic compounds, much like Atlantis® T3 HPLC columns
Waters is the Only Manufacturer Offering Two UPLC-Certified Particles

BEH Technology
In 1999, Waters launched the XTerra® family of HPLC columns featuring first generation hybrid particle technology (HPT). HPT enabled XTerra columns to become one of the most successful column products in the history of Waters. In HPT, the best properties of inorganic (silica) and organic (polymeric) packings are combined to produce a material that has:
· Superior mechanical strength
· Efficiency
· High pH stability and peak shape for bases.
The first generation methyl hybrid particles of XTerra columns, although very successful, did not possess the equivalent efficiency to that of a modern silica based product, or the mechanical strength to be useful for a UPLC type product. In 2005, a new second generation hybrid material was introduced which utilizes an ethylene bridged hybrid (BEH) structure. Compared to the first generation methyl hybrid particle of XTerra columns, the BEH particle of XBridge HPLC and ACQUITY UPLC BEH columns exhibit vastly improved efficiency, mechanical strength and pH range delivering a range of products ideally suited for flexible and efficient method development. BEH Technology is a key enabler behind the speed, sensitivity and resolution of UPLC separations.
The BEH Particle: One of the Key Enablers of UPLC Technology
USP L Listings
Choosing an HPLC column can be a very difficult process. The Chromatographic Packing Material (Particle “Brand Name” and Functional group) plays a major role in the success of a separation, both initially, when the method is first developed, as well as long term, if the method will need to be reproduced for many years.
The US Pharmacopeia (USP) is a standard source for many pharmaceutical methods. The USP specifies columns by packing materials rather than by manufacturer. Listed here are the recommended Waters HPLC columns suitable for most LC methods listed with the USP.
If you do not know the brand of column you need, and you would like to learn more about HPLC column particle technology packing material, including better ways to choose packing materials for your application needs, visit www.waters.com/selectivitychart.
Separation Modes
Reversed-Phase Chromatography
Reversed-phase chromatography is by far the most popular liquid chromatography separation technique employed in the laboratory today, and its popularity is reflected by the large selection of column products currently available from Waters.
In its simplest form, reversed-phase chromatography comprises of a polar mobile phase, typically mixtures of water or buffer with polar solvents such as methanol, acetonitrile or tetrahydrofuran, and a non-polar stationary phase such as a long-chain hydrocarbon bonded to a silica or hybrid support. In recent years, significant research and development effort has been targeted in the area of separation media aimed at improving column efficiency, pH stability and providing alternate selectivities.
Modern column chemistries from Waters can be broadly classified into three categories:
· Hybrid – Designed for maximum pH stability in order to provide the most flexible method development options. Available in XBridge™, ACQUITY® BEH, and XTerra® brands. pH range 1-12.
· Silica – Historically, the most commonly used reversed-phase support, SunFire™ and Symmetry® products are synthesized from raw materials in Waters Taunton manufacturing facility and are designed for maximum loadability. pH range 2-7
· Application-specific – Designed to target a specific application area, such as Atlantis® columns for enhanced retention of polar molecules
Normal-Phase Chromatography
Before the development of reversed-phase bonded phases, normal-phase chromatography was the most popular separation technique. It relies on the interaction of analytes with polar functional groups on the surface of the stationary phase, which is strongest when non-polar solvents are used as the mobile phase.
Normal-phase chromatography is a very powerful separations tool because of the wide range of solvents available that can be used to fine tune the selectivity of a separation. However, it has fallen into disfavor with many chromatographers because of some of the complexities involved. Under some circumstances, lengthy equilibration times or reproducibility problems may be encountered which are due largely to the sensitivity of the technique to the presence of small concentrations of polar contaminants in the mobile phase. If these problems are controlled, the technique typically gives chromatograms superior to reversed-phase methods due to the low viscosity of the commonly used solvents.
Normal-phase columns are available in the SunFire, Nova-Pak and Spherisorb product families.
HILIC (Hydrophilic-Interaction Chromatography)
Hydrophilic-interaction chromatography has been practiced for a long time, but the term HILIC has only recently come into common usage. Analytes are very polar compounds such as polar metabolites, carbohydrates or peptides.
HILIC can be viewed as an extension of normal-phase chromatography into the realm of aqueous mobile phases. The mobile phases are mixtures of water or buffer (< href="http://www.waters.com/webassets/cms/library/docs/usp_col_listings.pdf">here are the recommended Waters HPLC columns suitable for most LC methods listed with the USP.
If you do not know the brand of column you need, and you would like to learn more about HPLC column particle technology packing material, including better ways to choose packing materials for your application needs, visit www.waters.com/selectivitychart.
Separation Modes
Reversed-Phase Chromatography
Reversed-phase chromatography is by far the most popular liquid chromatography separation technique employed in the laboratory today, and its popularity is reflected by the large selection of column products currently available from Waters.
In its simplest form, reversed-phase chromatography comprises of a polar mobile phase, typically mixtures of water or buffer with polar solvents such as methanol, acetonitrile or tetrahydrofuran, and a non-polar stationary phase such as a long-chain hydrocarbon bonded to a silica or hybrid support. In recent years, significant research and development effort has been targeted in the area of separation media aimed at improving column efficiency, pH stability and providing alternate selectivities.
Modern column chemistries from Waters can be broadly classified into three categories:
· Hybrid – Designed for maximum pH stability in order to provide the most flexible method development options. Available in XBridge™, ACQUITY® BEH, and XTerra® brands. pH range 1-12.
· Silica – Historically, the most commonly used reversed-phase support, SunFire™ and Symmetry® products are synthesized from raw materials in Waters Taunton manufacturing facility and are designed for maximum loadability. pH range 2-7
· Application-specific – Designed to target a specific application area, such as Atlantis® columns for enhanced retention of polar molecules
Normal-Phase Chromatography
Before the development of reversed-phase bonded phases, normal-phase chromatography was the most popular separation technique. It relies on the interaction of analytes with polar functional groups on the surface of the stationary phase, which is strongest when non-polar solvents are used as the mobile phase.
Normal-phase chromatography is a very powerful separations tool because of the wide range of solvents available that can be used to fine tune the selectivity of a separation. However, it has fallen into disfavor with many chromatographers because of some of the complexities involved. Under some circumstances, lengthy equilibration times or reproducibility problems may be encountered which are due largely to the sensitivity of the technique to the presence of small concentrations of polar contaminants in the mobile phase. If these problems are controlled, the technique typically gives chromatograms superior to reversed-phase methods due to the low viscosity of the commonly used solvents.
Normal-phase columns are available in the SunFire, Nova-Pak and Spherisorb product families.
HILIC (Hydrophilic-Interaction Chromatography)
Hydrophilic-interaction chromatography has been practiced for a long time, but the term HILIC has only recently come into common usage. Analytes are very polar compounds such as polar metabolites, carbohydrates or peptides.
HILIC can be viewed as an extension of normal-phase chromatography into the realm of aqueous mobile phases. The mobile phases are mixtures of water or buffer (< href="http://www.waters.com/waters/nav.htm?cid=513226">GPC & GFC Columns Wide offering of GPC columns such as Styragel ideal for the separation of organic-soluble samples or, Ultrahydrogel best use to solve your specific problems in aqueous separations.
GPC & GFC Standards
Wide selection of calibration standards for organic and aqueous GPC/SEC., available as individual molecular weights, or in kits.
EMPOWER
Simplify the way you collect and report chromatography test results with Empower™ 2 Software. Empower 2 is Waters’ flagship chromatography data software (CDS) package for advanced data acquisition, management, processing, reporting, and distribution.
Simplify Data Acquisition, Processing and Reporting
Designed for a data-secured regulatory lab environment, Empower 2 will help your lab perform more efficiently and securely:
· Store and retrieve chromatography data using an embedded, relational database
· Calculate more precise peak integrations with ApexTrack™ algorithm
· Easily control Waters ACQUITY UPLC® and Alliance® HPLC systems as well as third-party HPLC and GC systems
· Achieve more enhanced detection capabilities when using Waters mass detectors or the Waters Photodiode Array (PDA) Detector
· Grow seamlessly from a single workstation to an enterprise-wide system with Empower 2’s scalable, modular architecture
Options That Match the Way You Work
With Empower 2 Software, you can multi-task with ease – no need for multiple software packages. Four intuitive interfaces help anyone in your lab work more efficiently. Choose the option that matches the way you work, and you’re up and running in just a few clicks:
· Acquire data and control a variety of instruments, including HPLC, UPLC®, and GC
· Apply advanced detection techniques such as MS and PDA without outsourcing to a third party
· Deploy applications such as dissolution, method validation, integrated chemical structures and polymer analysis
Unique Interface Options
Empower™ 2 Software represents a fundamental shift in UPLC™, HPLC, and LC/MS system usability. Our CDS software is equipped with unique interface options designed for your lab – regardless of the task or user’s skill level.
When users log in, they receive their assigned default interface option or, if authorized, they can select the interface most appropriate for their current task.
Unique Interface Options:
· QuickStart – Collect, process and report data from a single-window access. Use a wizard to set up methods and run assays. Navigate the entire Empower 2 environment from one screen, including instrument control, view plotted data in real time, view raw data, and access tools to customize reports.
· Pro - access to all software functions for system administrators or anyone who needs total control.
· Open Access - Select your method and the number of samples you need to run, and get started with the click of a mouse.
· Web user interface- easy access to information for remote or mobile users on the Internet or an intranet.
Empower's QuickStart interface

MassLynx Mass Spectrometry Software
The challenges of MS and MS/MS analyses are often compounded by the use of several types of analytical instruments and high personnel turnover. To maintain – and even increase laboratory productivity, turn to Waters software to simplify interaction with your MS system and retain the ability to perform advanced experiments.
Waters MassLynx™ Software improves your MS system with its intuitive interface, intelligent instrument control, and software features built around the focus of your analysis: the sample. With much of its development driven by input from our extensive user base, MassLynx has evolved into a powerful software package that delivers the versatility and flexibility you require.
Transform Data Into Usable Results
To assist with both data acquisition and the transformation of data into usable results, MassLynx Software offers easy-to-use Application Managers that allow you to focus the power of MS on your laboratory's specific tasks.
MassLynx MS Software
A fundamental platform to acquire, analyze, manage, and share mass spectrometry information
Quanpedia
Quanpedia is an extensible and searchable database for quantitative LC/MS and LC/MS/MS method information. Quanpedia simplifies and accelerates quantitative UPLC/MS/MS analytical method creation.
QCMonitor
QCMonitor for MassLynx Software automates “on time” quantitative data quality monitoring to determine whether a QC or blank sample is within tolerances specified by the user.










How many types of hplc columns?
There are several column types, according to their function, they can be classified as: a) Normal phase In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample. Therefore, the stationary phase is usually silica and typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these. b) Reverse phase In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water. c) Size exclusion In this column type, molecules are separated according to size. Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules. d) Ion exchange In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase. Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites. This HP 1090 Chromatograph is also equipped with an oven in the column compartment. The function of the oven is to provide an homogeneus air-bath temperature when it is required for some methods, such as the carbohydrate separation method which requires a constant temperature of 85ºC.

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