Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Investigation of Protein Structure
Chromatography
Gas-liquid chromatography method

Gas-Liquid Chromatography (GLC) is one of the most advanced, accessible, and efficient chromatographic techniques. The stationary phase in GLC is a solid, granular, porous support impregnated with a liquid sorbent. Therefore, implementing gas-liquid chromatography requires three components: a solid support, a liquid stationary phase, and a gaseous Mobile phase, known as the eluent.

Solid Support

Specially prepared diatomaceous earths are commonly used as Supports for packed columns in GLC. The surface of these Materials can be modified in various ways. The best results are achieved by washing with strong mineral acids to remove Metal Ions, followed by surface deactivation using silanizing Reagents. After such Treatment, the support surface is chemically more inert than the original material. Today, the commercial range of solid supports for GLC is vast, meeting virtually any demands of research laboratories. For the analysis of biological materials, various Chromosorbs (such as Chromosorb W) are most frequently employed.

Stationary Phase

In principle, almost any substance that can be anchored to a support and possesses low vapor pressure at the analysis Temperature can serve as a stationary phase in GLC. The commercially available range of stationary phase materials is simply immense. In routine biological research, silicones (such as the XE series — XE-30, XE-60) are most commonly used as relatively non-polar liquid phases. Polyesters (e.g., BDS — butane-1,4-diol succinate, NPGG — neopentyl glycol glutarate, EGA — Ethylene glycol adipate, PEGA — polyethylene glycol adipate, and many others) occupy a polarity range between silicones and polyglycols. The latter represent relatively polar stationary phases. In addition, various surface-active agents, organometallic compounds, and other substances can be utilized.

Mixtures of different substances are also frequently used as liquid stationary phases in GLC.

Mobile Phase

The mobile phase in GLC is typically an inert gas (nitrogen, helium, hydrogen, argon). For specialized purposes, other gases or vaporized substances (Water, ammonia, etc.) may also be used.

Basic Concepts

> Chromatographic curve (chromatographic peak) — the curve representing concentration (or any parameter proportional to concentration) at the Column outlet as a function of time from sample injection, or the volume of eluent passed through the column. An ideal chromatographic peak should have the shape of a regular Gaussian curve (an isosceles triangle). In practice, however, the peak shape is frequently distorted either at its front or rear edge (so-called peak tailing or fronting). The causes of peak broadening are quite diverse and will not be discussed here.

> Retention time (t_R) — the time elapsed between sample injection and The Emergence of the component concentration maximum from the column.

> Gas hold-up time (t_m) — the retention time of an absolutely inert substance (one that is not retained by the stationary phase). Finding such a substance is sometimes very difficult. Most often, air (if a catharometer detector is used) or methane (with a flame ionization detector) serves this purpose.

> Chromatographic peak width (ω) — the segment length measured between the intersection points of the baseline with tangents drawn through the inflection points of the ascending and descending Branches of the chromatographic peak. Peak width is frequently characterized by the length of a segment drawn parallel to the baseline between the ascending and descending branches at half of the peak height.

> Peak height (h) — the distance from the baseline to the maximum point of the chromatographic curve.

> Peak area — the area enclosed between the chromatographic curve and the baseline. This area can be determined in various ways: calculated using the triangle formula (if the peak is symmetrical), divided into small squares or rectangles, or by cutting out peaks plotted on paper and weighing them, etc. Modern chromatographs are typically equipped with electronic integrators that calculate peak areas automatically. The meaning of these parameters is illustrated in the figure below:

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Substance Retention Parameters

> Adjusted retention time (t'_R) — the component retention time minus the gas hold-up time (see figure):

t'_R - t_R - t_m

The values of t_R and t'_R strongly depend on the conditions and operating parameters of chromatography; therefore, they are rarely used to characterize the retention of a substance by the stationary phase. Retention volumes are less sensitive to analytical conditions.

> Specific retention volume (V_r) — the product of the retention time and the volumetric flow rate (v) of the mobile phase (carrier gas) at the column outlet:

V_R = t_RV

> Dead volume (V_m) — the retention volume of an absolutely inert substance that is not adsorbed on the stationary phase:

V_m = t_RV

> Adjusted retention volume (V’_R) — the difference between the volumes V_R and V_m:

> Adjusted retention volume (V_N). The value of V_R depends on the mobile phase flow rate. In GLC, this is accounted for using the James-Martin compressibility factors (j)

V_n = jV_R

> The column capacity factor (k’) is defined by the ratio:

k' = (t_R - t_m)/t_m

Parameters characterizing the Separation of two substances

У Relative retention (a) of substances 1 and 2 is defined by one of the following ratios:

а = (t'_R)₂(t'_R)₁ = k₂’/k₁’ = (V_R')₂/(V_R')₁

The value of a depends on the type of stationary phase and temperature, while showing little dependence on the nature, flow rate, and pressure of the carrier gas.

> Kovats retention index (I). One of the most practically important retention parameters, introduced by Kovats. Due to their intuitive nature, simplicity of determination, and low sensitivity to analytical conditions, these indices are tabulated for numerous substances and stationary phases. The index I is determined relative to two standard normal alkanes (1 and 2) between whose peaks the peak of the analyte is located (see fig.)

The value of I_x for a substance (X) whose peak lies between the peaks of two normal alkanes (1 and 2) (see fig.) is calculated using the formula:

where Z is the number of carbon atoms in the first alkane.

In addition to Kovats indices, attempts have been made in GLC practice to introduce more complex indices calculated from the retention times of 3 to 10 reference compounds. Although more accurate, they require significant effort to determine and have not found widespread use.

Degree of separation. The degree of separation of two adjacent peaks on a chromatogram is evaluated by the equation:

In quantitative analysis, the degree of separation must be at least 1.5. If the heights of two adjacent peaks differ significantly, ∆_1,2 must be even higher.