The Role of Ultrahigh Resolution Chromatography in the Chemical

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The Role of Ultrahigh Resolution Chromatography in the Chemical Industry H. MICHAEL WIDMER and KARL GROLIMUND Department of Analytical Research, CIBA-GEIGY Ltd., CH-4002 Basel, Switzerland

Trace investigations are routine activities of the industrial analyst, who attempts to solve problems in quality control, product research and development, and supports production, safety and marketing managers. Challenged by corporate specifications and requirements from authorities, his goal is to find economic methods of low detection limits and improved separation efficiencies. Instrumental techniques are increasingly combined with chromatographic methods in which low effluent flows and high separation power are pre-requisites. The number of impurities present in a sample at ppb level is orders of magnitudes larger than that present at ppm levels. Accordingly, there is a need for ultrahigh resolution chromatography techniques. Theoretical and practical aspects of ultrahigh resolution gas chromatography and high performance liquid chromatography are discussed. Examples from capillary GC and microbore HPLC are presented. A n a l y t i c a l chemistry i s an extremely dynamic branch o f science and the i n d u s t r i a l a n a l y s t i s c o n s t a n t l y faced with problems a s s o c i a ted with methodological changes. Only about 20 years ago t r a c e i n v e s t i g a t i o n s i n the ppm range became a c c e s s i b l e , today the ppb range i s under a t t a c k . T h i s e v o l u t i o n i s not simply a matter o f extending a n a l y t i c a l experience t o a f i e l d o f higher s e n s i t i v i t y and lower d e t e c t i o n l i m i t s , but i t i n v o l v e s the i n t r o d u c t i o n and development o f new separation techniques and new d e t e c t i o n methods. Furthermore i t i s accompanied by an appropriate change of mind and mental a t t i t u d e . U l t r a h i g h Resolution Chromatography

i n Industry

In our days the i n d u s t r i a l a n a l y s t s f i g h t on two f r o n t i e r s a t the 0097-6156/ 84/ 0250-0199$06.75/ 0 © 1984 American Chemical Society Ahuja; Ultrahigh Resolution Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

200

ULTRAHIGH RESOLUTION CHROMATOGRAPHY

same time. On one s i d e they are i n t e r e s t e d i n the development of low d e t e c t i o n l i m i t systems and on the other they s t r i v e to c r e a t e the b a s i s f o r high r e s o l u t i o n s e p a r a t i o n techniques. Both problems are i n t e r r e l a t e d and one cannot solve one problem without touching the other. The lower the c o n c e n t r a t i o n of a component i n a sample the l a r g e r i s the number of i n t e r f e r i n g substances i n t h i s sample. Matrix e f f e c t s are the most p e r s i s t e n t problems i n the d a i l y work of the i n d u s t r i a l a n a l y s t concerned with t r a c e and u l t r a t r a c e i n v e s t i g a t i o n s . T h i s i s demonstrated i n "Figure 1". I t represents a set of chromatograms i n which gasol i n e i s analyzed by c a p i l l a r y GC. The d i f f e r e n c e of the four chromatograms l a y s i n the change of s e n s i t i v i t y of the flame i o n i z a t i o n d e t e c t o r s e t t i n g . The top chromatogram i s taken with an a t tenuation of 4096, the l a s t with 1 . The f i g u r e shows that there i s one component present a t a c o n c e n t r a t i o n higher than 10 %, however, there are 10, 43 and 105 sample components a t concentrat i o n s higher than 1 %, 1000 ppm and 100 ppm, r e s p e c t i v e l y ("Figure 2"). This trend i s a most general one and not s p e c i f i c f o r gasol i n e . S i m i l a r r e l a t i o n s e x i s t with c i g a r e t smoke, p o l l u t e d a i r , water and ocean p o l l u t i o n as w e l l as i n d u s t r i a l products. There are s e v e r a l reasons f o r the i n d u s t r i a l a n a l y s t to show an i n t e r e s t i n u l t r a t r a c e i n v e s t i g a t i o n s and t h e r e f o r e he must cope with u l t r a h i g h r e s o l u t i o n chromatography. In chemical i n d u s t r y there i s a great awareness about the q u a l i t y of the products and q u a l i t y c o n t r o l has never been taken so s e r i o u s l y and consequently as i n our days. Unwanted s i d e e f f e c t s of c e r t a i n components, the presence of i m p u r i t i e s with c a r c i n o g e n i c , mutagenic or t e r a t o g e n i c e f f e c t s , corporate and governmental requirements f o r c e the q u a l i t y c o n t r o l people to apply t r a c e and u l t r a t r a c e methods. A s i g n i f i c a n t d r i v e f o r the need to lower d e t e c t i o n l i m i t s stems from o c c u p a t i o n a l s a f e t y and h e a l t h c o n s i d e r a t i o n s and r e quirements. Hazardous chemicals such as dimethyl s u l f a t e may be used as educts i n the s y n t h e s i s of i n d u s t r i a l goods, others such as e p i c h l o r o h y d r i n e or b i s ( c h l o r o m e t h y l ) e t h e r may be generated d u r i n g a chemical process. "Table I" summarizes the maximum permissable working concent r a t i o n s of these substances i n a i r . In Switzerland (CH) and Germany (GFR) they are c a l l e d MAK-values, i n the USA they are known as Threshold L i m i t Value-Time Weighted Average (TLV-TWA). CIBAGEIGY e s t a b l i s h e d i t s own P e r m i s s i b l e I n t e r n a l Exposure L e v e l (PIEL) f o r substances, such as b i s ( c h l o r o m e t h y l ) e t h e r , f o r which o f f i c i a l l e v e l s are not a v a i l a b l e . Monitors. There are s e v e r a l techniques to check and c o n t r o l hazardous m a t e r i a l i n the a i r of working areas. In recent years the permanent s u r v e i l l a n c e of ambient a i r has become an important i s sue of the s a f e t y and h e a l t h people i n i n d u s t r y and the a n a l y s t has to conform with these needs and provide the appropriate methods and instrumentation.

Ahuja; Ultrahigh Resolution Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

WIDMER AND GROLIMUND

Figure 1.

Role in the Chemical Industry

Gasoline analyzed by c a p i l l a r y GC

(SE-54, 60


m).


Constituent Concentration Figure 2. P l o t of c o n s t i t u e n t number versus c o n s t i t u e n t concentration i n g a s o l i n e .


13.


Table I.

203

Maximum P e r m i s s i b l e Exposure L e v e l f o r DMSj ECH and BCME

Species USA TLV-TWA 100 ppb 2 ppm 1 ppb

DMS ECH BCME


Exposure Level GFR CH MAK MAK 10 ppb 10 ppb 3 ppm 5 ppm 1 ppb

PIEL 500 ppt

Monitor Detection Limit 2 ppb 100 ppb 200 ppt

In our company we developed a v e r s a t i l e and f l e x i b l e monitor system shown i n "Figure 3". This p a r t i c u l a r instrument i s used f o r the s u r v e i l l a n c e o f b i s ( c h l o r o m e t h y l ) e t h e r i n ambient a i r . Our aim was t o design and c o n s t r u c t a mobile automated s y s tem, performing survey analyses a t a r a t e of 1-5 analyses per hour on the hour."Figure 4" shows the o p e r a t i o n a l p r i n c i p l e of the instrument. I t c o n s i s t s of compact module u n i t s , which may be arranged f o r s p e c i f i c a p p l i c a t i o n s and serve d i f f e r e n t needs (1_, 2). The a i r sample i s trapped on an adsorbent, then thermally desorbed and gas chromatographed, followed by an appropriate de t e c t i o n system, such as FID, FPD, ECD o r a mass spectrometer. Examples o f d e t e c t i o n l i m i t s are given i n "Table I " . Theoretical

Considerations

The chromatographic r e s o l u t i o n i s given by the column e f f i c i e n c y and the s e l e c t i v i t y o f the chosen separation system. Efficiency.

The e f f i c i e n c y may be d e s c r i b e d by "Equation 1"

(1 )

For packed columns the e f f i c i e n c y i s given by the q u a l i t y and par t i c l e s i z e o f the column packing. For w a l l coated c a p i l l a r i e s the e f f i c i e n c y i s a matter of the c o a t i n g f i l m p r o p e r t i e s . Further more, the e f f i c i e n c y depends on the i n j e c t i o n mode, s o l v e n t e f f e c t s , flow r a t e and column dimensions. Generally, the column e f f i c i e n c y i s expressed i n terms o f the t h e o r e t i c a l p l a t e number Ν o r the height equivalent t o a theoretical plate, Η : The p l a t e number i s

Ν = 16 (

16 Ε

and the height equivalent to a t h e o r e t i c a l p l a t e .


(2)

204


Figure 3.

B i s ( c h l o r o m e t h y l ) e t h e r monitor.

Sample Probe

Detector N2

Column

I Adsorption Tube jQ

2 ^ IT

^jj^ Recorder

Heating Wire ^Magnetic Valve Transfer Line

À Pump

Figure 4.

Schematic view of monitor ( o p e r a t i o n a l p r i n c i p l e ) .


13.



205

For a given column a t optimum flow c o n d i t i o n s the maximum e f f i c i e n c y and t h e o r e t i c a l p l a t e number can be c a l c u l a t e d , as w e l l as the minimum H. "Table I I " and "Table I I I " g i v e some r e p r e s e n t a t i v e values f o r HPLC and c a p i l l a r y GC, r e s p e c t i v e l y . Table I I .

E f f i c i e n c y o f HPLC Columns with D i f f e r e n t Materials 10 cm Column

Diameter o f Packing M a t e r i a l i n ym

E

Max

N

18 25 32

10 5 3

Max

5,000 10>000 16,000

20 cm Column Ν Max Max

E

Packing

Min mm 0.02 0.01 0.006

10,000 20,000 32,000

25 35 45

Golay (3>, 4) introduced a s i m p l i f i e d formula f o r the minimum height e q u i v a l e n t t o a t h e o r e t i c a l p l a t e f o r w a l l coated c a p i l l a r i e s , given i n "Equation 4" (j>) 1/2 1 + 6k' + 11k' H

Table I I I .

3(1 + k' )

E f f i c i e n c y o f Wall Coated C a p i l l a r y Columns

Inner Diameter mm 0.10 0.20 0.30 0.40 0.50

(4)

Min

H Mm mm

N /m Max

0.084 0.168 0.252 0.337 0.421

11,880 5,950 3,960 2,970 2,380

w

N .-/m eff

8,250 4,130 2,750 2,060 1 ,650

Η err mm 0.121 0.242 0.364 0.485 0.606

The height equivalent t o a t h e o r e t i c a l p l a t e may be c a l c u l a t e d by a semi-empirical Van Deemter equation (£) H

=

A

+

~ u

+

C ·û

(5)



206

In HPLC the Van Deemter curves, r e l a t i n g H with the mean l i n e a r v e l o c i t y of the mobile phase, are h y p e r b o l i c and the minimum H i s obtained with r e l a t i v e l y low l i n e a r v e l o c i t y around 1 and 2 mm/s. A d e v i a t i o n from the optimum flow i s connected with a pronounced increase of the height e q u i v a l e n t , according to the Β and C terms of "Equation 5", r e s p e c t i v e l y . In SFC t h i s increase i s l e s s dramatic, because the d i f f u s i o n c o e f f i c i e n t i s 2 to 3 orders of magnitude l a r g e r than i n HPLC. As a consequence the minimum Η becomes almost indépendant of the flow r a t e over a l a r g e range of l i n e a r v e l o c i t y . For 3 ym columns a l i n e a r flow v e l o c i t y between 0.4 and 1.2 cm/s or 2.8 to 8.5 ml/min f o r a column of 4.6 mm inner diameter may be achieved with a p l a t e number around 120,000 per meter. Selectivity. The s e l e c t i v i t y , c h a r a c t e r i z i n g the e l u t i o n of d i f f e r e n t substrates i s described by "Equation 6".

two

(6a)

A,Β

I t depends mainly on the d i f f e r e n c e of the i n t e r a c t i o n s between the substrates and the c a r r i e r m a t e r i a l and the mobile and s t a t i o n a r y phases. Introducing the separation f a c t o r and c a p a c i t y f a c t o r one obtains

s

. 1°-ΐΔ1

a

)

(

a

A, Β

1 + k

(6b)

B

Resolution. The r e s o l u t i o n i s d e r i v e d from the e f f i c i e n c y s e l e c t i v i t y according to "Equation 7" R and

s

=

Ε

Β

. S

and

(7a)

Α,Β

therefore R

s

= (-^) 4

.

(-2=1) α

1 +k '

)

(7b)

Β

Conclusions. From the foregoing c o n s i d e r a t i o n s we can make some conclusions about the p o s s i b i l i t i e s to improve the chromatographic r e s o l u t i o n . In i n d u s t r y e f f o r t s are undertaken to improve the e f f i c i e n c y as w e l l as the s e l e c t i v i t y . They are summarized i n the following l i s t s .


13.



207

Ways to Improve the S e l e c t i v i t y : - t o enhance s p e c i f i c i n t e r a c t i o n s of the s u b s t r a t e with the mobile phase by • gradient elution • ion pairing • precolumn d e r i v a t i s a t i o n . m o d i f i e r s i n SFC - to enhance s p e c i f i c i n t e r a c t i o n s of the substrate with the s t a t i o n a r y phase by • multidimensional chromatography • column switching • s p e c i f i c phases (such as i n s i z e e x c l u s i o n chromatography or o p t i c a l isomer separation) • precolumn d e r i v a t i s a t i o n - to choose s p e c i f i c d e t e c t i o n systems (there i s a p a r t i c u l a r need f o r new and improved HPLC d e t e c t o r s ) - hyphenated systems • post column d e r i v a t i s a t i o n Ways t o Improve the E f f i c i e n c y : - The e f f i c i e n c y depends on the column q u a l i t y and t h e r e f o r e on • column packing • particle size • f i l m p r o p e r t i e s of w a l l coated columns - column dimensions G e n e r a l l y , the expert chromatographer r e l i e s on h i s own column packing techniques, because commercial packed columns f o r HPLC do not always provide optimum e f f i c i e n c y , but are l e s s v u l n e r a b l e to the treatment of l e s s experienced chromatographers. Practical

Aspects

Chromatographic t h e o r i e s concerned with e f f i c i e n c y and s e l e c t i v i ty are g e n e r a l l y based on homologous compounds, f o r which s i m i l a r temperature dependant p r o p e r t i e s are known. However, i n chemical i n d u s t r y the a n a l y s t i s r a r e l y concerned with mixtures of homologous substances. In these samples the temperature e f f e c t s may outweigh those of d o u b l i n g the f i l m thickness and column length of c a p i l l a r y columns. The f o l l o w i n g c a p i l l a r y GC t e s t s were performed with the Grob Test Mixture (2), l i s t e d i n "Table IV". I t represents a comb i n a t i o n of c o n s t i t u e n t s of v a r i a b l e p o l a r i t y and r e f l e c t s q u i t e w e l l the r e a l world of i n d u s t r i a l problems. In "Figure 5" a comparison i s made between isothermal and temperature programmed c a p i l l a r y GC with equal r e t e n t i o n times f o r the l a s t e l u t i n g component of the Grob Test Mixture. In order to achieve these c o n d i t i o n s the isothermal chromatogram was run at 150 C and the temperature programmed chromatogram was s t a r t e d at 60 C f o r 1 min, followed by a temperature r a t e of 4 C/min, whereby peak A e l u t e s at 197°C.



Figure 5. Comparison of isothermal and temperature grammed c a p i l l a r y GC of a t e s t mixture.


pro-

13.

Table IV.

Grob Test Mixture

Compound

Solvent

209



Chemical Formula

-

Constituents

Abréviation

Peak I d e n t i fication i n "Figures 5, 6, 8 and 11"

-

M-R

D

L Κ

1-Octanol

10 22 C H OH

10 ol

I

1-Nonanal

C H CHO

al

G

2,6-Dimethyl Phenol

(CH ) C H OH

Ρ

F

11

H

A

Ε

2,3-Butanediol Decane

H

4 10°2

C

Undecane

H

8

8

C,j g-Methylester -Methylester

17

17

3

C

2,6-Dimethyl A n i l i n e

C^

C

2

6

3

H

11 24 (CH ) C H NH 3

C

2

6

H

3

2

( a

10 19°2 V H 0 (CH )

C l 1

2 1

2

D i c y c l o h e x y l Amine

C

12 23

C.j -Methyl es t e r

C

12 23°2

H

H

3

N

( C H

3

)

10e

D

11e

C

am

Β

12e

A

C e r t a i n l y d i f f e r e n t s e l e c t i v i t i e s and e f f i c i e n c i e s are obser ved with the two chromatograms. The r e s o l u t i o n i s f a v o r a b l e i n the temperature programmed e l u t i o n f o r the v o l a t i l e compounds, the contrary i s true f o r the l e a s t v o l a t i l e substances. E f f e c t s o f F i l m Thickness. "Figure 6" shows three chromatograms of the Grob sample mixture. A l l columns were 1 5 m long and coated with the s t a t i o n a r y phase SE-54. The temperature g r a d i e n t was 1 C/min s t a r t i n g a t 60 C. From top t o bottom the column had a f i l m thickness of 0.5, 1 .0 and 2.0 ym, r e s p e c t i v e l y . A f i r s t i n s p e c t i o n o f the chromatograms r e v e a l s t h a t the c o lumn with the t h i n n e s t f i l m had the best o v e r a l l r e s o l u t i o n , de monstrated by the base l i n e r e s o l u t i o n o f peaks F, G and H. A more d e t a i l e d look shows that with i n c r e a s i n g f i l m thickness the e f f i c i e n c y i n c r e a s e s , but a t the same time the s e l e c t i v i t y de creases. This i s p a r t i c u l a r l y so f o r the separation o f peaks Β and C. However, the r e s o l u t i o n i s increased f o r v o l a t i l e substan ces when the f i l m thickness i s increased. With r a t h e r t h i c k f i l m columns i t i s p o s s i b l e t o separate the main c o n s t i t u e n t s of earth gas as i s demonstrated i n "Figure 7". E f f e c t s o f Column Length. The i n f l u e n c e of column length on the r e s o l u t i o n and e f f i c i e n c y i s known. To increase the r e s o l u t i o n o f a homologous substance p a i r by two, we must increase the column length by a f a c t o r o f f o u r .


210





SE-54 , 60 m, 2.0 μπι, 20 °C

Cigaret Lighter

Butane 6as

Earth Gas Ethane

Butane

Butane

Methane

Il Propane

Propane Propane

Pentane

Butane

Pentane

Figure 7. Separation of c i g a r e t l i g h t e r , e a r t h and butane gas with t h i c k f i l m c a p i l l a r y columns.


212


The s i t u a t i o n may be q u i t e d i f f e r e n t f o r non-homologous con s t i t u e n t s . "Figure 8" represents the chromatograms o f SE-54 c a p i l l a r y colums a l l coated with a 0.5 ym f i l m . The length of the c o lumns were from top t o bottom 15, 30 and 60 m, r e s p e c t i v e l y . Con s i d e r i n g the l a s t two peaks A and C, one observes separation num bers o f 27, 39 and 55 f o r the top, middle and bottom chromatogram, r e s p e c t i v e l y , as expected from theory. Substrates A and C are i n deed members o f a homologous s e r i e s . But f o c u s s i n g on the performance o f the 30 m column, one r e cognizes that peaks F, G and H are not r e s o l v e d . Apparently, con s t i t u e n t s F and G have s i m i l a r r e t e n t i o n times. Increasing the column length t o 60 m i s not s a t i s f a c t o r y e i t h e r . However, one observes a true b a s e - l i n e r e s o l u t i o n with the s h o r t e s t column on top. A r a t h e r unexpected r e s u l t from e f f i c i e n c y theory, but a common case i n the i n d u s t r i a l world. Obviously, sub s t r a t e s F and G have a d i f f e r e n t dependence o f t h e i r r e l a t i v e r e t e n t i o n times on a change o f temperature ( f o r explanation see a l s o "Figure 12"). Peak Inversions. Peak i n v e r s i o n s are q u i t e common i n c a p i l l a r y gas chromatography. In "Figure 9" peak i n v e r s i o n i s achieved by se l e c t i n g d i f f e r e n t temperature r a t e s with the same column. In "Figure 10" the i n v e r s i o n i s observed based on columns with d i f f e r e n t f i l m t h i c k n e s s . These examples demonstrate that i n many cases a b e t t e r chromatographic separation i s obtained t a k i n g advantage of f a v o r a b l e temperature e f f e c t s . These are best ex p l a i n e d by a c o n s i d e r a t i o n o f r e t e n t i o n i n d i c e s . "Figure 11" represents the simultaneous e f f e c t s o f changing f i l m thickness and column l e n g t h . These examples a l s o demonstrate that there are techniques a v a i l a b l e t o produce c a p i l l a r y columns of high p r e c i s i o n and r e p r o d u c i b i l i t y ( 8 ) . The columns are charac t e r i z e d from top t o bottom by a f i l m thickness of 2.0, 1.0 and 0.5 μπι and a column length o f 15, 30 and 60 m, r e s p e c t i v e l y . Kovats I n d i c e s . The Kovats Index o f a substance X, r e f e r r i n g t o a chromatogram run a t temperature Τ and with a s t a t i o n a r y phase Ρ i s c a l c u l a t e d according t o "Equation 8"

log

log

mX V m, Β t' m. A

T,P

T,P T,P ) + I Β Β

(8)

nvΒ Β and A r e f e r t o the alkanes e l u t i n g before and a f t e r substrate X.




4->

CP

•

β

0

c

rH

X

70

Η

00

13.


219


Improved Detector Systems - tandem d e t e c t o r s such as ECD/FID i n GC - e l e c t r o c h e m i c a l d e t e c t o r s i n HPLC - o p t i c a l sensors (optrodes) - chemical f i e l d e f f e c t transducer (chemfet) - laser-based d e t e c t o r s (e.g. photoacoustic d e t e c t o r s , l a s e r - i n duced f l u o r e s c e n c e , e t c . ) Hyphenated Systems - GC/TEA - HPLC/TEA - HPLC/MS - SFC/MS - SFC/FT-IR I n t e r d i s c i p l i n a r y Influence from other A n a l y t i c a l Techniques - flow i n j e c t i o n a n a l y s i s - e l e c t r o c h e m i c a l development - l a s e r technology - biochemical and enzyme immobilization techniques These tendencies are i l l u s t r a t e d with an example from microbore column LC/MS. "Figure 13" d e s c r i b e s the LC/MS i n t e r f a c e used and gives a schematic view of the i n t e r f a c e p r i n c i p l e . "Figure 14" r e presents the mass spectrum of an analyzed sample, i n v o l v i n g a com pound with a molecular weight of 530. Conclusions The s t a t e of the a r t i n u l t r a h i g h r e s o l u t i o n chromatography allows the i n d u s t r i a l a n a l y s t already now to introduce t h i s technique i n h i s d a i l y work. However, f u r t h e r improvements are needed to make f u l l use of the inherent p o t e n t i a l i n i n d u s t r i a l a p p l i c a t i o n s . Legend of Symbols Chromatographic e f f i c i e n c y E f f i c i e n c y of peak A and B, r e s p e c t i v e l y Height e q u i v a l e n t to a t h e o r e t i c a l p l a t e (HETP) Minimum HETP

Min ef f T,P I, I

E f f e c t i v e HETP

n

Kovats index of substrate X at temperature Τ and i n g to the separation phase Ρ

refer-

Capacity f a c t o r Number of t h e o r e t i c a l p l a t e s f o r column chromatography Ν

,N

Maximum and e f f e c t i v e N, r e s p e c t i v e l y



Kovats Index

SE-54

1130 2,6-0iraethyl Phenol

y y

1120 1110 1-Nonanal 1100

Undecane

1090 1080 l-0ctanol

1070

-L

50

70 90 110 130 Elution Temperature (°C)

150

Figure 12. Temperature dependence of Kovats i n d i c e s f o r 2,6-dimethyl phenol, 1-nonanal, undecane and 1-octanol.

Reagent Gas

COIIMI

Effluent

Figure 13· Schematic o f microbore LC/MS i n t e r f a c e .


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The Role of Ultrahigh Resolution Chromatography in the Chemical

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