Chapter 1

A Tutorial-based Theory Course in Chromatography

Based on C.F. Poole and S.K. Poole “Chromatography Today”, Elsevier: Amsterdam, 1991.

The tutorial form requires active students, in order to obtain a dialogue, instead of a monologue on the

part of the tutor. The book chosen for this course is well suited for this kind of studies, since it touches

many aspects of the phenomenons discussed. A dialogue is, however, only possible if the students are well

prepared for each tutorial. You are therefore urged to read the material listed below and in the separate

reading list before arriving at the tutorial classes.

The following is a list of the text from the book which should be read prior to each tutorial, as numbered

in the course schedule.

Tutorial # Book chapter

1 1

2 2

3 3

4 4.1 … 4.4

5 4.5

6 4.6 … 4.8

7 5, 6 and 7 (see tutorial problems for reading instructions)

8 8.1 … 8.9

9 8.10 … 8.16

10 “Choosing the Correct Separation Techniques”© Copyright, Knut Irgum, 1996-2000. All rights reserved. Page 2

Tutorial Problems for Poole and Poole: “Chromatography Today”, Chapter 1

1. Discuss the basic foundation and various modes of chromatography, as outlined on page 2 ff.

2. How does the compressibility of gases complicate the measurements of retention volumes in GC?

3. Gas chromatography can be used to obtain certain thermodynamic data. How can this be used to

strengthen the proof of identity for an eluting analyte compound?.....................................................[8]

4. The number of theoretical plates, n, is most frequently determined parameter from measurements of

peak width and retention time. How can this measure of column efficiency be determined from only

the peak height and area of a Gaussian peak? ...................................................................................... [11]

5. When is it relevant to use “Trennzahl” (TZ; also known as “separation number”, SN) instead of the

number of theoretical plates for measuring column efficiency in gas chromatography?.................. [14]

6. Discuss the causes of band-broadening in packed and open tubular column chromatography?. [16 ff.]

7. Choice of column diameter and length, and stationary phase type and film thickness affect the separation in open tubular gas chromatography. How are these parameters related to the main goals of

an analytical separation: Maximum separation at minimum time and cost in simplest possible system.

[23] ................................................................................................................................................................

8. Identify some anomalies that typically lead to tailing peaks in chromatography. ............................. [26]

9. Statistical moments are formed by successive integrals of a Gaussian peak. What uses can be made of

statistical moment of various degree in evaluating chromatographic peaks? .................................... [27]

10. Explain the physical rationale for using the exponentially modified Gaussian (EMG) function when

evaluating chromatographic peaks. ...................................................................................................... [28]

11. Consider equation 1.47, and identify the three fundamentally different processes that contribute to

providing resolution (typically the prime optimisation criterion) in chromatography.................... [30]

12. Among the options available for improving resolution, which ones are typically most effective

considering the main optimisation goal stated above?........................................................................ [33]

13. Is there any value of discussing the term “peak capacity” (PC) when assessing the suitability of a

particular set-up for a real analysis situation?...................................................................................... [35]

14. Elucidate the difference between thermodynamic and kinetic means of affecting a chromatographic

separation process.................................................................................................................................. [36]

15. Confer Table 1.8 and explain why capillary columns are more efficient than packed columns in gas

chromatography. ................................................................................................................................... [37]

16. Which parameters are important when the separation time is to be minimised in GC? Can the

techniques described in the book be used also for other chromatography modes?...................... [45-48]

17. What are the implications on the separation process, if a GC column outlet is connected directly to

the vacuum source of a mass spectrometer? ........................................................................................ [46]

18. Disregarding linear dynamic range of the detector, the sample capacity of a chromatographic

method is limited both by the lowest detectable amount and the maximum sample capacity of a

column. How is the minimum detectable amount related to band-broadening in the column, and

how is the maximum peak concentration related to properties of the analyte and the separation

column in gas chromatography? Finally, assess the utility of Qs

, as defined in equation 1.61. .... [48-50]

19. Saxton has introduced the concept “emergence temperature” as a tool aiding in positively identifying

a compound eluting in temperature programmed GC. Discuss the value of this approach........ [56-57]

20. Serially connected column of different selectivity have been proposed as a powerful technique for

optimising separations in HRGC. What are the tools used for developing such techniques, and what

difficulties are involved in the developmental procedure? Discuss also Figure 1.13. ................... [58-59]© Copyright, Knut Irgum, 1996-2000. All rights reserved. Page 3

21. Below which particle size is there a dramatic decrease in the mass transfer limitation in the mobile

phase in liquid chromatography? ......................................................................................................... [65]

22. On page 66-67 it is suggested that large bore columns are used to alleviate the problems arising from

the use of highly efficient separation materials in HPLC. Is this a sound solution to the problem? [66]

23. Describe the causes and possible effects of the temperature inhomogeneities that can occur in long

LC columns operated at high pressure. ................................................................................................ [69]

24. Extended length LC columns are discussed on page 70. Are such columns practical? What is the

problem associated with preparing long LC columns with good efficiency, and how can it be

overcome? .............................................................................................................................................. [70]

25. What is the principle advantage of miniaturising an HPLC system, and what instrumental problems

are connected with a transition from conventional columns to miniature systems? Describe a way to

determine if an existing system is fit for use with miniature systems................................................. [74]

26. Reduced parameters have been introduced as a way of comparing separation efficiency of separation

columns independent of scale. What are the advantages of using this approach?............................. [76]

27. Discuss Tables 1.18 and 1.19, considering limitations that may prevent utilisation of some of the

breathtaking efficiencies listed in the Table 1.18. ........................................................................... [81-82]

28. Column temperature is frequently controlled in HPLC. What are the main reason for doing so, and

is there typically much to be gained by increasing (or decreasing) the column temperature? Can

problems arise from thermostatting the column at a temperature different from ambient?............ [84]

29. Ventilate the concept static vs. dynamic noise in chromatographic detector. Does this distinction

provide a clue to troubleshooting a malfunctioning HPLC system? .................................................. [86]

30. Signal anomalies in chromatographic detectors are characterised as short and long term noise, and

drift. Which of these spurious signals is most troublesome to the chromatographer? ..................... [87]

31. Elaborate on “dynamic range” and “linear range”........................................................................... [88-89]

32. Calibration on chromatography can take place through external or internal standards, or by standard

additions. Discuss advantages and drawbacks for each of these variants. .......................................... [91]© Copyright, Knut Irgum, 1996-2000. All rights reserved. Page 4

Tutorial Problems for Poole and Poole: “Chromatography Today”, Chapter 2

1. Discuss the intermolecular forces that give rise to retention in chromatography ........................ [107-8]

2. Review the requirements for an optimal support in packed gas chromatography. ......................... [119]

3. Why is it necessary to silanize supports and column tubes in gas chromatography, what reactions are

used, and how can we test if the procedure has been successful? ..................................................... [123]

4. What precautions should be taken to protect the stationary phase in a gas chromatography column

from deterioration?.............................................................................................................................. [125]

5. What are the interactions probed by the individual probe compounds in the Grob test?.............. [163]

6. How do different open tubular columns differ with respect to stationary phase breakdown? ....... [168]

7. Under what conditions is surface adsorption of analyte at the gas-liquid interface most likely to

occur in a gas-liquid chromatography system?.................................................................................. [171]

8. Study Figure 2.11. What does it show? ............................................................................................... [174]

9. List the criteria that must be fulfilled for a compound to be suitable for use as a stationary phase in

gas chromatography. ................................................................................................................[108 and 184]

10. What limits the lower and upper useful temperatures for a GC stationary phase? ......................... [184]

11. What are the two different criteria listed for determining maximum temperature for a GC stationary

phase? ................................................................................................................................................... [184]

12. How precise are the notions polarity and solvent strength?.............................................................. [185]

13. Account for the free energy of solution of a methylene group as a means of representing the polarity

of a GC stationary phase...................................................................................................................... [187]

14. The McReynolds retention index is afflicted with several flaws which has rendered the substantial

amount of data collected with this technique nearly useless. Account for these flaws. ................... [190]

15. How useful is the dendrogram in Figure 2.13? .................................................................................. [195]

16. Compare Table 2.11 and Figure 2.13. Can the same pattern be seen?...................................[187 and 195]

17. Gas-solid chromatography (GSC) has a number of disadvantages that have limited the popularity of

the technique. There are……. ............................................................................................................ [199]

18. In which particular applications is GSC the technique of choise? .................................................... [200]

19. What are the principal retention mechanisms for silica and alumina GSC packings? How does water

affect the retention on these sorbents, and why does this effect arise? ............................................. [201]

20. Visualise some analytical settings where selectivities such as those displayed in Figures 2.15 and 2.16

could be useful. .........................................................................................................................[203 and 205]

21. Review Table 2.15 and identify compounds that could hardly be separated by other chromatographic

techniques than GSC. .......................................................................................................................... [207]© Copyright, Knut Irgum, 1996-2000. All rights reserved. Page 5

Tutorial Problems for Poole and Poole: “Chromatography Today”, Chapter 3

1. Discuss why a better overall system performance is required when open tubular columns are used,

compared to packed columns?................................................................................................................[#]

2. Gases used as carrier, as fuel and as makeup gases, and for pneumatic purposes in GC may contain

impurities that are deleterious on the column and system hardware. What kind of contaminations

are most problematic and how are they dealt with? ..............................................................................[#]

3. The carrier gas flow in the separation column can be controlled by either flow or pressure. What are

the advantages and drawbacks of each of these ways of administering the mobile phase? .................[#]

4. The normal temperature operating range for a column oven is 40 to 350 °C. Try to describe the effect

of an oscillating or spacially inhomogeneous oven temperature..........................................................[#]

5. The basic requirement for an sample injection in chromatography is not to degrade the separation

potential of the column. Try to describe the ideal injection! What major conflicts do arise?..............[#]

6. What major factors influence the accuracy of syringe injections? ........................................................[#]

7. Injections in GC are nowadays done by autoinjectors that operate with very high precision. What

autoinjector parameters should be controlled and optimized by the operator?..................................[#]

8. There are several different injections techniques available for open tubular columns. Why are packed

column systems less sensitive for the type of injection technique?.......................................................[#]

9. Septum bleed is an often encountered problem. What does it mean and how do you detect it? ........[#]

10. The split injector is really an isothermal vaporizing injector. Describe the principle and hardware

configuration. ..........................................................................................................................................[#]

11. Try to figure out what happens in the syringe/injector/column assembly during a split injection. ...[#]

12. What parameters influence the actual split ratio ? What are the major negative effects of a high split

ratio, and what are the positive effects? ..................................................................................................[#]

13. Study Table 3.2 on page 246 and make sure you can find it easily later!..............................................[#]

14. Under what circumstances is splitless injection an attractive alternative to split injection?.................[#]

15. Describe some experiments suitable for optimizing the conditions of a splitless injection. ...............[#]

16. Peak compression (or refocusing) is an expression commonly discussed in chromatography. Why is

refocusing needed with splitless injection and what are the basic mechanisms? .................................[#]

17. Explain the expression broadening in time of eluting peaks. How can you avoid the effect and even

gain important advantages?.....................................................................................................................[#]

18. Why does the programmed temperature vaporization (PTV) injectors show less discrimination and a

better split ratio/flow ratio relationship?................................................................................................[#]

19. Explain the term retention gap and when is it preferred for on-column injections. Discuss with respect

to the flooded zone phenomenon and peak compression. .......................................................................[#]

20. Band broadening in space is a problem encountered with on-column injection. What are the general

guidelines for an successful on-column injection..................................................................................[#]

21. What is the fundamental advantage of a) a general, and b) a specific detector in GC?.......................[#]

22. Suppose you have recorded a chromatographic peak at a certain carrier flow rate with a concentration

sensitive detector. What will be the effect on peak area and peak height if the flow rate is doubled?

What would be the effect if a mass sensitive detector was used instead?...............................................[#]

23. What is the basic principle of an ionization detector? ............................................................................[#]

24. What are the prerequisites for obtaining a signal in the flame ionization detector(FID)? ....................[#]© Copyright, Knut Irgum, 1996-2000. All rights reserved. Page 6

25. The N and P sensitive detector (thermionic sensitive detector TID) is similar to the FID, but bears

some important differences for its function. Try to describe the most important aspects. ................[#]

26. What are the benefits of the low ionization efficiency and the flexible ionization energy of a

photoionization detector?..........................................................................................................................[#]

27. Why is the electron capture detector (ECD) referred to the “easiest to use but least understood”! .....[#]

28. What is actually detected in the ECD? In what way is the ionization efficiency important?...............[#]

29. Why are pulse sampling techniques used with ECD’s? .........................................................................[#]

30. What factors give rise to detection selectivity with the ECD? ...............................................................[#]

31. Discuss the advantages and drawbacks of different GC detectors with respect to sensitivity and

selectivity. What detector will be both the most sensitive and the most selective?..............................[#]