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Introduction to Chromatography Separation of a mixture of components A and B by column elution chromatography. Partition in Chromatography Stationary phase, mobile phase, & analyte form a ternary system. Using the Partition Coefficient: Plate Theory Column divided into theoretical pieces (plates).
Definitions I V R, Retention Volume – Volume required to carry a band of component molecules – Primary quantity, but hard to measure! T 2 > t 1" title="Van Deemter Model “B” Term Illustration of band broadening due to molecular diffusion.
Optimum Mobile Phase Velocity How would this plot differ for packed columns versus capillary columns? Gas Chromatography – Overview Sample is vaporised and injected onto head of a chromatography column.
Sample Injection GC column efficiency requires that the sample be of suitable size (to prevent column over loading) and be introduced as a plug of vapour. Sample Valve Injection (a) Sample mode (b) Inject mode Sample valves are convenient for on-line gas stream analysis. 0.01 ? l of a liquid sample is preferred volume - a precision syringe for this volume is both expensive and fragile.
Pyrolysis Gas Chromatography (PGC) A version of reaction chromatography in which a sample is thermally decomposed to simpler fragments before entering the column. Cyanobacteria are phototrophic prokaryotes, which inhabit environments of widely differering conditions in nearly all habitats on earth. So they are able to survive in freshwater as well as in sea water by adjusting the water potential of the cytoplasm. Sensing of salinity changes and fine tuning of de novo synthesis and uptake of compatible solutes in the moderate halotolerant model strain Synechocystis sp.
By the in vitro analysis of enzyme activities under defined conditions we can monitor the dependency on different parameters as well as the interaction of proteins by biochemical methods. Additional regulatory factors can be identified by affinity chromatography with overexpressed proteins or by transposon mutagenesis and subsequent selection of positive clones.
The participation of proteases in osmoregulation is under investigation by the characterization of FtsH protease mutants.
Degradation of proteolytic substrates can be analysed in vivo by the 2D-Page technique and in vitro after heterologous production of putative substrates and incubation with inverted membrane vesicles containing different FtsH proteases or after reconstitution of the membrane bound proteases.
By the subsequent mutagenesis of proteases as well as target proteins the mechanisms of substrate recognition and degradation will be studied on the molecular level.
Identification of mechanisms involved in stress dependent activation and inactivation of enzyme activities during the osmotic acclimatization process in Synechocystis.
Identification and characterization of new components involved in regulatory principles and pathways and verification of occurence in other cyanobacteria or eubacteria. In all living cells the water content of the cytoplasm has to be adjusted in relation to the external medium osmolality.
In Synechocystis the synthesis and accumulation of the osmoprotective compound glucosylglycerol (GG) is an essential process during osmotic acclimatization. GG synthesis is performed by the enzymes Glucosylglycerol-phosphate-synthase (GGPS), which is the key enzyme and Glucosylglycerol-phosphate-phosphatase (GGPP). These enzymes might form a complex after the release of the GgpS enzyme from a postulated inhibitor (A). During steady state regulation most of the enzyme is inactivated by a so far unknown mechanism (A), but can be reactivated rapidly by an increase of the external salt concentration.

We aim to understand the mechanism of GG synthesis regulation by searching for involved proteins by transposon mutagenesis and screening for clones with deregulated GG synthesis. By heterologous production of the involved enzymes (B) we analyze activation and inactivation by in vitro enzyme assays and focus also on enzyme kinetics and structure.
The proposed model for GG synthesis regulation (A) includes the release of the GgpS enzyme from an inhibitor and the interaction with the GgpP enzyme during GG synthesis. By a so far unknown mechanism the complex is inactivated but can be reactivated after an increase of the external salt concentration. For in vitro investigation involved components are heterologously produced (B) and analyzed by enzyme assays under different conditions (B). The structure function relation of the GgpS enzyme can be visualized by the proposed structure model.
Protein quality control and degradation of misfolded and irreversibly inactivated enzymes is an essential process in all living cells. A ubiquitous proteolytic machinery is the membrane bound ATP and Zn dependent FtsH protease complex, constituted of six FtsH proteins. While in heterotrophic bacteria only one gene encoding an FtsH protease is present cyanobacteria possesses four different FtsH proteins.
We have identified the FtsH2 protease of Synechocystis as a player in osmoregulation responsible for the degradation of irreversibly inactivated GgpS enzyme. This is an essential procedure because the ftsH2 mutant is impaired in GG synthesis and therefore salt sensitive (A). After heterologous production and purification of substrates the degradation can be analyzed in vitro (C). By a combination of physiological and biochemical approaches we want to understand regulatory mechanisms on the molecular level.
Besides the cultivation of Synechocystis on agar plates or in Erlenmeyer flasks (A) growth with elevated CO2 concentrations can be performed (B). Cell size and shape under osmotic stress conditions can be analyzed by light (C) or fluorescence microscopy. For generation of mutants we use insertions of antibiotic resistance markers (A) or deletions of genes (B). For screening procedures random mutant collections are generated by transposon mutagenesis (D). All routine molecular biology techniques for cloning and modification of genes (A) are established.
Gene expression can be monitored by Northern-Blot as well as Western-Blot techniques (C) and in collaboration with other groups by DNA microarray (D) or 2D-PAGE analysis (E).
For identification of regulatory compounds and quantification of external and internal substrate concentration we use HPLC, GC as well as GC-MS.
By the time dependent substrate concentration analysis the kinetic parameters of the transport process can be determined. Spectroscopic methods are in use for the analysis of pigmentation (A) the efficiency of photosystems as well as protein conformations (C) and protein protein interactions. Data can be visualized by computer based structure predictions (C) in order to design new modifications, e.g.
The ChromaBLOGraphy series continues for the use of internal standards with medical cannabis potency testing by GC-FID (I’ve listed the first two parts in the series immediately below as web-links).  This third part demonstrates the positive impact an internal standard can have on quantitative accuracy. To be able to explain how partition of an analyte between stationary phase and mobile phase effects separation. In position (a) the stream to be sampled flows through a loop of calibrated volume while the carrier gas alone passes through the column.

1993, 65, 827 IUPAC Compendium of Chemical Terminology Many non-volatile solids can be decomposed thermally to produce characteristic gaseous products that can be chromatographed.
2: Working model of the regulation of osmolyte synthesis in Synechocystis on the posttranslational level. To be able to identify and explain the factors influencing chromatographic separation in terms of resolution and specificity. The effectiveness of a column in separating two analytes depend in part on the relative rates at which the two components are eluted. Separation is based upon the partition of the analyte between a gaseous mobile phase and a liquid phase immobilised on the surface of an inert solid (GLC) at a temperature above boiling point of analyte (multi-analyte: temperature programming). The syringe technique is most common and can be used with both gas and low viscosity liquid samples by inserting the needle through a rubber septum to the column inlet port. Mostly glass liners – zero dead volume The sample volume is then split between the column and the split outlet.
In position (b) the loop is placed in the carrier gas stream and the entrapped sample is swept along to the column.
Samples are placed directly on a small coil of Pt wire where it can be heated to several hundred degrees in a few milliseconds while the carrier gas is flowing over it. After an initial salt stress dependent activation the GgpS protein forms a complex with the second enzyme of the pathway GgpP, which results in GG-synthesis. Strain PCC 6803 encoding glucosyl-glycerol-phosphate synthase is involved in osmolyte synthesis. Identify the factors influencing different sample injection techniques and be able to discuss the advantages and disadvantages of each type. These rates are determined by the magnitude of the equilibrium constants for the components partioned between the stationary phase and mobile phase. The region into which the needle projects must be heated in order to flash vaporise the sample. Sample valves are becoming more prevalent for quantitative work employing both liquids and gases to introduce a reproducible volume of sample onto a column. The essential GG synthesis is performed by the key enzyme of the pathway GgpS and the second enzyme GgpP. PCC 6803: The compatible solute synthesizing enzyme GgpS is one of the targets for proteolysis.
Identify the factors influencing different analyte detection systems and be able to discuss the advantages and disadvantages of each type. Peak broadening due to zone (eddy) diffusion resulting from multitude of pathways a molecule can pass through column.
GC columns are either packed (with silica particles coated in stationary) or capillary in nature.
If this injector is operated in split mode, the amount of sample reaching the column is reduced (to prevent column overloading) and very narrow initial peak widths can be obtained. For maximum sensitivity, the injector can be used in so-called splitless mode, then all of the injected sample will reach the column.

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