The Grob Test

The most comprehensive test for capillary columns is the Grob test (K. Grob Jr. G. Grob, and K. Grob, J. Chromatogr. 156 (1978) 517; K. Grob, G. Grob, and K. Grob Jr., J. Chromatogr. 219 (1981) 13.). It must be beared in mind that a lot of specific tests exist but that the comparison of specific tests with a general test is wrong.

The importance of the Grob's Test is based on the comprehensive information on column performance that can be gathered from the chromatogram. Because of its unique characteristics it can be used to follow eventual changes in column condition and , with some restrictions, for column comparison, based on:

• Standardization

  This is the heart of the test, since all the information it affords is temperature-dependent (flow dependent). Temperature programing and carrier flow are individually optimized, according to column length and nature of carrier. This is a fundamental step for determination of separation efficiency.
  

• Activity (Adsorption, Acidity and/or Basicity)

  This is based on measurement of percent peak height for test compounds (1-octanol ol, D(-)-2,3-butanediol D, 2,6-dimetylaniline A, 2,6-dimethylphenol P, 2-ethylhexanoic acid S, dicyclohexilamine am, and very seldom n-nonanal al) relative to a 100% line defined by peak tops of hydrocarbons (n-decane 10 and undecane or dodecane 11, and methyl esters (methyl decanoate E₁₀, methyl undecanoate E₁₁, and methyl dodecanoate E₁₂).
  

• Use of esters instead of alcanes for determination of separation efficiency

  This is a superior method since retention of esters is less dependent on column polarity in striking contrast with alcanes.
  In the Grob test, separation efficiency is accessed by calculation of the Separation Number (Trennzahl TZ) introduced by Kaiser and defined as the resolution of two consecutive ester peaks differing by one methylene unit according to:
  TZ= [(Tr _E11-Tr _E10)/W_{h/2 E11}-W_{h/2 E10})]-1

  The same calculation is made for the pair E₁₁-E₁₂ and the TZ is calculated as the mean for the two values. The Trenzahl is related to Resolution by
  

  TZ= (R/1.177)-1

  and to the Kovats' Retention Index dI
  

  TZ= (100/dI)-1 In practice TZ the effective peak number, i.e. the number of peaks that can be separated between the two homologues.

• Column type and film thickness

  For each column type the test delivers a typical elution profile (please refer to the above publications of Grob et col.). The difference of measured elution temperature of E₁₂ and the standard temperature may be used to calculate film thickness according to a Nomogram included in the original publications of Grob et col.

  Glass vs. fused silica tubing

  In spite of the widespread popularity of the fused-silica tubing we should not forget the classical glass capillaries. The instinctive thought that fused-silica is a superior material for the construction of capillary columns is by no means correct. With exception of an eventual easier manipulation and in some instances easier deactivation, there are no significant differences between both type of capillaries. On the other side, the number of liquid phases amenable to fused-silica coating is more limited. Modern coatings have a thermal stability that is higher thean the thermal stability of the exterior polyimide coating of fused-silica capillaries. This is significant in high temperature GC.

  Comparison between FS and Glass Capillaries

  Factors	Glass	Fused-silica
  Chromatographic quality	Identical	Identical
  Surface treatment	Important	Moderate
  Applicable coatings	Unlimited	Limited
  Thermostability	Very high	Limited
  Installation	easy	Easiest
  Coupling ability	Good	Very good
  Price	Can be home-made	High
  Transparency	Optimal	None

  Test Your Column

• Chromatographic profile for an inert efficient non-polar column (methyl silicone)

  If your test looks like this you are off the hook (The elution pattern is different for other phases- see referenced work)
  

• Strong Activity yet Good Resolution

  If you've got this:
  

  You are in trouble

  The column shows an exceptional high activity: strong adsorption of ol and al, moderate acidity (P and am). The esters show some interaction with the column.

  REMEDY:However, the efficiency (TZ= 23) still allows the use of the column for separation of non-polar compounds.

• Good inertness but poor resolution

  

  REMEDY:You have good a good inert column but with a very poor resolution (TZ= 10). You still may use it if you do not have resolution problems.

• Deposition of involatiles

  A- On the initial part of the column

  

  Your column has been used for analysis of "dirty" samples, for instance, samples of biological origin. The samples contain less volatile components. Although they evaporate in the injector, they recondense in the initial part of the column and accumulate there. The column shows excessive "bleeding" and adsorptive properties. The integrity of the liquid film, however, is untouched as shown by the elution temperatures of the esters.

  REMEDY: Cut some 3-4 initial spires. Rerun the test. The initial performance should be reestablished.

  B- On the injector

  If you got this.
  

  You got a very dirty injector. The involatiles in the sample tend to deposit onto the glass insert and carbonized by continuous heating, leaving a very active surface.

  REMEDY: Replace the glass insert by a clean one and rerun the test. The initial performance should be reestablished.

• Acquired activity

  If you got this:
  

  You got activity that may have been acquired through injection of samples containing traces of detrimental residues. For instance, repeated work with acylated derivatives. This type of acquired activity is due to a number of causes, all of them related to the presence of active groups within the column: impurities or reappearing of active silanol groups.

  REMEDY: Frequently the performance of the column can be restored by on-column silylation. A number of commercial silylation mixtures exist for such purpose. On-column silylation helps to cover exposed active silanol groups and to form silylated volatile derivatives of otherwise non-volatile impurities accumulated within the column. Silylate and rerun the test. The initial performance should be restored.

• Effect of rectification on glass capillaries

  If you use glass capillaries and got this:
  

  The fact that glass is a rigid material requires that the extremities of a glass capillary be straighted up in order to fit in the oven. This operation is usually performed by careful heating with a flame of a micro-burner under a mild current of air. During this operation some liquid phase evaporates and burns out of the column by the action of the air flow. In the heating zone the coating is thus lost, exposing active points.

  REMEDY: With the help of a syringe and against a counter-flow of gas (an aquarium pump will do) inject a solution of 1% PEG 20 M in CH₂Cl₂ in the first 3 spires of the coil. Aliviate the pressure and let the solution reflux back intro the syringe. Repeat the operation some 10 times. Allow the solvent to evaporate. Recondition. Repeat the test. The initial performance should be restored.

  Alternatively do not use a flame but use an adequate electrical oven instead.

  Conclusion

  The full potential of capillary gas chromatography can not be attained only by purchasing an adequate instrument and installation of a most perfect column. The understanding of the chemical and physical peculiarities of the separation process and column anatomy are indispensable. The quality of results depends finally of column performance, which is susceptible to aggressions and aging. Quality of results depends upon the column, on its performance and maintenance. Periodic testing is therefore a must.

  The Grob test is a comprehensive test for assessment of column performance and performance comparison. In many situations, dramatic changes of chromatographic performance have simple causes that are easy to identify and correct saving from unnecessary discarding a still high valuable column. <

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