Pesticide Analytical Methodology - American Chemical Society

3 Performance Evaluation of Liquid Chromatographic Columns JOSEPH J. DESTEFANO

Downloaded by CORNELL UNIV on October 2, 2016 | http://pubs.acs.org Publication Date: October 30, 1980 | doi: 10.1021/bk-1980-0136.ch003

Ε. I. Du Pont de Nemours & Company, Inc., Instrument Products Division, Wilmington, D E 19898

The measurement of column performance c r i t e r i a to compare columns, column packings, and column l o a d i n g procedures has long suffered from the n o n - s t a n d a r d i z a t i o n of t e s t procedures and d i s agreement as to which of the many performance c r i t e r i a are most significant. I t i s the purpose of t h i s paper to p o i n t out s e v e r a l p r a c t i c a l aspects which must be considered when designing experiments to t e s t column performance. When one attempts to evaluate LC columns, the f i r s t question that must be answered i s , "What i s good?". In other words, which of the many measurable performance parameters are most s i g n i f i c a n t i n terms of the u l t i m a t e use of the column—chromatographic analysis? Most experts i n HPLC recognize the value of measuring the reduced column performance parameters, such as reduced p l a t e height (h) and column r e s i s t a n c e parameter ( φ ) , because these dimensionless parameters make comparisons of columns much e a s i e r ( 1 ) . Therefore, experts i n chromatography might define a good column as one which had a reduced p l a t e height of between 2 and 5 at the minimum of a reduced p l a t e height versus reduced l i n e a r v e l o c i t y plot. This p l o t would r i s e very gently from the minimum as v e l o c i t y increased. In a d d i t i o n , s p e c i f i c p e r m e a b i l i t y or column r e s i s t a n c e parameter values would be c o n s i s t e n t with t h e o r e t i c a l values f o r the p a r t i c l e s i z e of the packing and mobile phase viscosity. On the other hand, most users of HPLC columns might define a good column as one which separates t h e i r p a r t i c u l a r sample mixture i n r e l a t i v e l y short a n a l y s i s times, one that remains s t a b l e for a s i g n i f i c a n t l y long p e r i o d of time, and one which, at the end of i t s l i f e t i m e , can be r e a d i l y replaced with a column of s i m i l a r performance.

0-8412-05 81 -7/80/47-136-031 $05.00/0 © 1980 American Chemical Society Harvey et al.; Pesticide Analytical Methodology ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Both of these d e f i n i t i o n s of column "goodness" have t h e i r s t r e n g t h s . The e x p e r t s d e f i n i t i o n has the important advantage of being able to compare r e s u l t s obtained from columns c o n t a i n i n g d i f f e r e n t packings, packed i n d i f f e r e n t ways, u s i n g d i f f e r e n t t e s t systems, w i t h the t e s t i n g being performed i n v a r i o u s l a b o r a t o r i e s . But the u s e r s d e f i n i t i o n i s r e a d i l y v i s u a l i z e d i n p r a c t i c a l terms which r e l a t e d i r e c t l y to the use of the column i n the i n d i v i d u a l l a b o r a t o r y . The manufacturers of HPLC columns must of n e c e s s i t y , be s e n s i t i v e to the needs of the chromatographers, but that s e n s i t i v i t y should not mean that q u a n t i t a t i v e column e v a l u a t i o n techniques can be ignored. Therefore, the two d e f i n i t i o n s of column "goodness" can be combined and s i m p l i f i e d to d e s c r i b e the f o l l o w i n g performance c r i t e r i a : 1


1

• • • • •

Column e f f i c i e n c y Peak symmetry Column p e r m e a b i l i t y Column s t a b i l i t y Column r e p r o d u c i b i l i t y

I t must then be decided which of the v a r i o u s measures of these c r i t e r i a , shown i n Table I , are the most g e n e r a l l y u s e f u l . COLUMN EFFICIENCY The most commonly used c r i t e r i o n f o r judging column performance i s e f f i c i e n c y as measured by the number of t h e o r e t i c a l p l a t e s or column p l a t e count (N) e x h i b i t e d by the column d u r i n g the s e p a r a t i o n of a t e s t mixture. The l a r g e r the number of t h e o r e t i c a l p l a t e s , the more l i k e l y i t i s that the column w i l l produce the d e s i r e d s e p a r a t i o n s . However, w h i l e popular, Ν i s not a complete performance parameter f o r making comparisons. For example, Ν does not take i n t o account p a r t i c l e s i z e as does the reduced p l a t e h e i g h t , h. Another measurement, hmin, accounts f o r a l l of these f a c t o r s as w e l l as the mobile phase l i n e a r v e l o c i t y and sample d i f f u s i o n . However, Ν i s the term most commonly recognized as being r e l a t e d to r e s o l u t i o n ( 2 ) , as shown i n Equation 1:

Resolution

« ±

j

a

~

g '

+

k

,j

< JT]

(Eq. 1)

Hence, r e s o l u t i o n , a key c r i t e r i o n f o r HPLC column u s e r s , i s maximized f o r a given mobile p h a s e / s t a t i o n a r y phase system by maximizing N. Therefore, even i f researchers are i n c l i n e d to measure h ^ ^ as t h e i r column e f f i c i e n c y parameter, manufacturers and users of LC columns can be f o r g i v e n f o r c o n t i n u i n g to use Ν f o r i n t e r n a l l a b o r a t o r y purposes.

Harvey et al.; Pesticide Analytical Methodology ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Harvey et al.; Pesticide Analytical Methodology ACS Symposium Series; American Chemical Society: Washington, DC, 1980. • • • •

Column e f f i c i e n c y

QUANTITY

(HETPJ

Solute r e t e n t i o n ( k , capacity factor) S e l e c t i v i t y (alpha) Packing c h a r a c t e r i s t i c s ( i e . , s u r f a c e area of adsorbents, s u r f a c e coverage of bonded phases, e t c . )

1

Long-term t e s t i n g Accelerated testing

Column S t a b i l i t y

• • •

Specific permeability Column r e s i s t a n c e parameter Pressure r e q u i r e d f o r g i v e n f l o w and mobile phase Flowrate achieved w i t h g i v e n pressure and mobile phase

Column P e r m e a b i l i t y

Column R e p r o d u c i b i l i t y

Skew Tau/Sigma r a t i o Asymmetry f a c t o r

Peak Symmetry

T h e o r e t i c a l p l a t e s (N) 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 Reduced p l a t e height (h; Minimum reduced p l a t e h e i g h t (h ) min

MEASURED

PERFORMANCE PARAMETER

PERFORMANCE CRITERIA

TABLE I



34

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ANALYTICAL

METHODOLOGY

P l a t e number by i t s e l f i s not a s u f f i c i e n t l y r e l i a b l e s p e c i f i c a t i o n . The p l a t e numbers e x h i b i t e d by a column can be v a r i e d by a d j u s t i n g the t e s t c o n d i t i o n s which are u s u a l l y designed to maximize those v a l u e s . For example, very low v i s c o s i t y mobile phases such as pentane are o f t e n used i n s p i t e of t h e i r p r a c t i c a l d i f f i c u l t i e s such as high v o l a t i l i t y to take advantage of the s u p e r i o r mass t r a n s f e r p r o p e r t i e s of such s o l v e n t s . A l s o , t e s t s o l u t e s o f t e n are chosen f o r t h e i r low p o l a r i t y to minimize band t a i l i n g , and f o r t h e i r low molecular weight to maximize mass t r a n s f e r e f f e c t s r a t h e r than f o r t h e i r r e l a t i o n s h i p to r e a l - l i f e samples. In a d d i t i o n , the t e s t i s o f t e n conducted at unusually slow f l o w r a t e s to take advantage of the f a c t that most column packings are more e f f i c i e n t under these c o n d i t i o n s . I t should be s t r e s s e d that there i s nothing wrong w i t h these p r a c t i c e s . In f a c t , column performance i s best compared under i d e a l thermodynamic c o n d i t i o n s (1). Hence, t e s t systems should be chosen to produce the best column performance, s i n c e most workers l i k e to see how w e l l a column r e a l l y can perform. However, i t should be recognized that when c o n s i d e r i n g the number of p l a t e s s p e c i f i e d f o r a column, i t i s necessary to examine the t e s t c o n d i t i o n s used to generate that number. PEAK SYMMETRY Another problem a s s o c i a t e d w i t h u s i n g the number of t h e o r e t i c a l p l a t e s (N) as a performance c r i t e r i o n i s that there are s e v e r a l equations which can be used to make t h i s c a l c u l a t i o n , as shown i n Figure 1. A l l of these equations are equivalent f o r gaussian peaks. However, f o r t a i l i n g peaks, a l l of the equations are subject to e r r o r , some more than o t h e r s . Therefore, a measure of peak symmetry i s r e q u i r e d to be able to determine the v a l i d i t y of p l a t e height measurements. Several methods can be used to measure the degree of t a i l i n g e x h i b i t e d by a peak. One method i s a computer-calculated value f o r the mathematical peak skew based on a novel peak a n a l y s i s method ( 3 ) . B r i e f l y summarized, Figure 2 shows that the contour of a peak can be described as having two components: a Gaussian component having the standard d e v i a t i o n , sigma, and an exponential m o d i f i e r having the time constant, tau. Peak t a i l i n g increases w i t h the tau/sigma r a t i o as shown i n Figure 3. This computer-measured r a t i o can be used to c a l c u l a t e peak skew (4_) u s i n g Equation 2: (Eq.

2)



3.

DESTEFANO

Performance

Evaluation

TANGENT METHOD

Ν = 16 ( t / t )

AREA METHOD

Ν = 2*(h't /A)

HALF-WIDTH METHOD

N= 5.54 ( t / W

R

w

z

R

R

2

1/2

)

2

of LC Columns

35

Figure 1. Equation for calculating the number of theoretical plates (number may vary with: mobile-phase viscosity-column temperature; test solute type and molec ular weight; mobile-phase velocity)

Figure 2. Graphical depiction of τ (ex ponential tailing component) and σ (gaussian component) features of an asymmet rical peak (τ/σ ratio related to peak skew)



Figure 3.

Computer-generated peaks having different skew (τ/σ) values (constant variable τ ) 2


3.

Performance

DESTEFANO

Evaluation

of LC

Columns

37


With previous methods, peak skew could not be determined w i t h s u f f i c i e n t p r e c i s i o n to be meaningful ( 3 ) . However, w i t h the tau/sigma method, peak skew i s e a s i l y c a l c u l a t e d t o give p r e c i s e , u s e f u l , q u a n t i t a t i v e i n f o r m a t i o n on peak shape. The tau/sigma method f o r c a l c u l a t i n g peak skew i s p r e f e r r e d over other methods s i n c e t h i s approach has a sound t h e o r e t i c a l b a s i s and values can be measured w i t h greater p r e c i s i o n and accuracy. However, u s i n g peak skew as a performance c r i t e r i o n does s u f f e r from one drawback: i t i s necessary to have a s o p h i s t i c a t e d computer to make the c a l c u l a t i o n . Therefore, most users of HPLC columns w i l l have t o use hand-calculated v a l u e s f o r peak asymmetry measurement (Figure 4 ) . However, these hand-calculated values can be c o r r e l a t e d t o a f i r s t approximation t o the computer-generated values of peak skew u s i n g the graph shown i n F i g u r e 5. COLUMN PERMEABILITY Measurement of column p e r m e a b i l i t y i s not c l e a r - c u t . The p e r m e a b i l i t y of a column can be determined i n s e v e r a l d i f f e r e n t ways. For example, i t can be measured as the s p e c i f i c p e r m e a b i l i t y , K° (EQ. 3 ) , which c o r r e c t s f o r mobile phase v i s c o s i t y and column length ( 5 ) : K° =

where:

ν η ΔΡ L

= = = =

ψ

mobile mobile column column

(EQ. 3)

phase l i n e a r v e l o c i t y (cm/sec) phase v i s c o s i t y (poise) pressure (atm χ 10&) l e n g t h (cm)

Because of these c o r r e c t i o n s , s p e c i f i c p e r m e a b i l i t y (K°) i s a measurement f o r making comparisons among columns of v a r i o u s types. U n f o r t u n a t e l y , i t i s d i f f i c u l t to convey what t h i s measurement means when one i s attempting to use a column f o r a given ap p l i c a t i o n . A more e a s i l y v i s u a l i z e d and more p r a c t i c a l way t o d e s c r i b e column p e r m e a b i l i t y i s t o s p e c i f y a f l o w r a t e measured f o r a given column w i t h a given mobile phase a t a given pressure and temperature. A l t e r n a t i v e l y , the pressure r e q u i r e d t o produce a given f l o w r a t e w i t h a given mobile phase and temperature can be s p e c i f i e d . These l a t t e r two s p e c i f i c a t i o n s are not a b s o l u t e measurements as i s the s p e c i f i c p e r m e a b i l i t y , but they do suggest to the column user the pressure r e q u i r e d t o c a r r y out a p a r t i c u l a r separation. COLUMN STABILITY The s t a b i l i t y of the column packed bed and the packing bonded


38

ANALYTICAL

METHODOLOGY


PESTICIDE

ASYMMETRY

Figure 5.

FACTOR

Graph of first-approximation relationship between peak skew and the peak asymmetry factor


3.

DESTEFANO

Performance

Evaluation

of LC

39

Columns

phase are d i f f i c u l t to measure. U s u a l l y , these parameters are measured through a c c e l e r a t e d column o p e r a t i o n t e s t s . These t e s t s normally u t i l i z e auto-samplers, computer data handling systems, high operating pressures and mobile phase f l o w r a t e s , and elevated temperatures. In a d d i t i o n , d i f f i c u l t mobile phases such as concentrated b u f f e r s c o n t r o l l e d at very high or very low pH values are o f t e n used to examine column s t a b i l i t y .


The r e s u l t s of such t e s t s are most u s e f u l f o r determining d i f f e r e n c e s i n s t a b i l i t y among columns from v a r i o u s manufacturers ( i e . , they are good r e l a t i v e t e s t s ) . However, these t e s t s should not be considered absolute measures of column s t a b i l i t y s i n c e t h e i r r e l a t i o n s h i p to r e a l - l i f e use c o n d i t i o n s i s not known. COLUMN REPRODUCIBILITIES L a s t l y , column-to-column r e p r o d u c i b i l i t y experiments must be designed to be s e n s i t i v e to v a r i a t i o n s i n p a r t i c l e s u r f a c e area and pore s t r u c t u r e , d i f f e r e n c e s i n the amount of coverage of bonded phases, and v a r i a t i o n s i n the packed bed which may r e s u l t during column l o a d i n g . The r e t e n t i v i t y ( c a p a c i t y f a c t o r , k ) of a t e s t s o l u t e under standard c o n d i t i o n s i s one i n d i c a t o r of column to-coluran r e p r o d u c i b i l i t y . This i s e s p e c i a l l y true f o r bondedphase packings s i n c e the amount and chemical c h a r a c t e r i s t i c s of the bonded-phase have l a r g e e f f e c t s on the r e t e n t i o n of a s o l u t e . This f a c t i s best i l l u s t r a t e d by the data presented i n Table I I . Three d i f f e r e n t batches of an experimental bonded-phase packing were prepared using three d i f f e r e n t s y n t h e t i c approaches. The r e s u l t i n g three packings contained about the same amount of organic m a t e r i a l , but the r e t e n t i o n of benzyl a l c o h o l on each packing was g r e a t l y d i f f e r e n t under i d e n t i c a l chromatographic c o n d i t i o n s . The point i s c l e a r : simply knowing that a packing i s "an o c t a d e c y l bonded phase with 15% o r g a n i c " i s not s u f f i c i e n t to determine whether column-to-column v a r i a t i o n s w i l l degrade a d i f f i c u l t separation. f

While r e t e n t i v i t y s p e c i f i c a t i o n s are u s e f u l , an even b e t t e r and more s e n s i t i v e t e s t f o r column-to-column r e p r o d u c i b i l i t y i s the s e l e c t i v i t y f a c t o r , alpha. T h i s f a c t o r measures the r a t i o of the r e t e n t i v i t y or c a p a c i t y f a c t o r s ( k ) of two s o l u t e s and determines i f there are any d i f f e r e n c e s between columns which would r e s u l t i n the two peaks moving c l o s e r together or f u r t h e r apart. 1

f

U n l i k e the r e t e n t i v i t y measure, k , r e l a t i v e l y minor changes i n alpha can have very major e f f e c t s on r e s o l u t i o n as shown i n Table I I I . The v a r i a n c e s i n alpha that can be t o l e r a t e d from column-to-column to maintain a given s e p a r a t i o n (R >1.25) i s dependent on the alpha value i n v o l v e d . Table IV stows these allowable v a r i a n c e s f o r s e v e r a l values of alpha. This t a b l e


40

PESTICIDE

ANALYTICAL

METHODOLOGY

TABLE I I

EFFECT OF BONDED PHASE ON RETENTION


Packing Lot

% Organic

k

1

of Benzyl A l c o h o l *

#1

13.7

4.7

#2

12.6

7.7

#3

13.5

11.2

*Conditions:

Bonded phase - 3 experimental batches of p r o p y l n i t r i l e , mobile phase - 2/98 isopropanol/hexane.

TABLE I I I

EFFECT OF k

FOR:

·

Constant

Ν

•

Constant

α

•

Varying

k

R R

FOR:

·

s s

for k

f

for k

T

Constant

•

Constant

•

Varying

R R

s s

f

Ν k

f

=

4900

=

1.24

f

AND α ON Rs

+ 1 0 % from 2.0

of 1.8 of 2.2

=

4900

=

2.0

= «

2.18 o r

. . 2.25 + 3.5%

o r

, , 2,16 + 44%

0

o c

2.33

α + 10% from 1.24

f o r α of 1.116 f o r α of 1.364

» 1.21

0

Λ

/ / a /

= 3.11



3.

Performance

DESTEFANO

Evaluation

of LC

41

Columns

TABLE IV

ALLOWABLE

VARIANCE IN ALPHA

ALPHA

ALLOWABLE VARIANCE*

1.2

6.7%

10.0%

1.3

13.8%

16.9%

1.4

20.0%

22.9%

*To m a i n t a i n R **To m a i n t a i n R

g

ALLOWABLE VARIANCE**

>1.25 assuming Ν = 4900 and k' = 2.0 >1.25 assuming Ν = 10,000 and k' = 2.0


42

PESTICIDE

ANALYTICAL

METHODOLOGY


demonstrates that the s p e c i f i c a t i o n of a column s e l e c t i v i t y f a c t o r w i t h i n narrow l i m i t s can i n d i c a t e i f the d i f f i c u l t s e p a r a t i o n achieved on one column w i l l be accomplished on another s i m i l a r column, assuming constant N. There i s a p r a c t i c a l d i f f i c u l t y , however, i n d e s i g n i n g s e l e c t i v i t y t e s t s which w i l l provide t h i s k i n d of i n f o r m a t i o n . I t i s d i f f i c u l t to f i n d two s o l u t e s which a r e s u f f i c i e n t l y d i f f e r e n t so that t h e i r r e t e n t i v i t i e s w i l l r e a c t d i f f e r e n t l y t o changes i n column c o n d i t i o n s but which w i l l s t i l l e l u t e f a i r l y c l o s e t o one another i n a s i n g l e t e s t run. G e n e r a l l y , i t i s best to use t e s t compounds w i t h d i f f e r i n g f u n c t i o n a l groups to maximize the chances of the t e s t system being t r u l y d i a g n o s t i c . A f i n a l important r e p r o d u c i b i l i t y s p e c i f i c a t i o n should be considered which a p p l i e s s p e c i f i c a l l y to bonded-phase packings. F i r s t , the bonded-phase should be s p e c i f i e d as being polymeric or monomeric. I f p o l y m e r i c , i n f o r m a t i o n on the % organic o r % carbon for the packing and the chemical s t r u c t u r e of the bonded phase should be provided. However, as shown before, t h i s i n f o r m a t i o n i s o f t e n not s u f f i c i e n t to determine l o t - t o - l o t chromatographic r e p r o d u c i b i l i t y . I f the bonded phase i s monomeric, data on the % organic or % carbon and chemical s t r u c t u r e a r e a l s o u s e f u l , but i n a d d i t i o n , the surface coverage c a l c u l a t e d from these v a l u e s (6) should a l s o be provided (EQ. 4 ) . Coverage = (ymoles/m^)

where :

M

^

xlO

6

(EQ. 4)

W =

weight of organic l a y e r (g/g packing)

M =

molecular weight of bonded group (g/Mole)

S =

the s p e c i f i c surface area of the adsorbent, c o r r e c t e d f o r the weight of bonded phase (m /g packing) 2

Coverage values should be maintained constant from packing l o t to packing l o t t o s t a n d a r d i z e the number of unreacted a c i d i c s i l a n o l groups s t i l l present on the s i l i c a s u r f a c e . These a c t i v e , a c i d i c groups may i n t e r a c t w i t h s o l u t e s t o cause a mixed r e t e n t i o n mechanism. This e f f e c t w i l l c o n t r i b u t e s i g n i f i c a n t l y t o peak t a i l i n g , e s p e c i a l l y f o r b a s i c s o l u t e s . Because of s t e r i c c o n s i d e r a t i o n s , i t i s not p o s s i b l e t o r e a c t a l l of the s i l a n o l groups w i t h the s i l a n e reagent. However, by maximizing the number of groups r e a c t e d , the remaining s i l a n o l s a r e o f t e n made i n a c c e s s i b l e f o r i n t e r a c t i o n w i t h most s o l u t e s because of s t e r i c s h i e l d i n g . Therefore monomeric bonded phases, w i t h maximum, r e p r o d u c i b l e s u r f a c e coverage a r e p r e f e r r e d to assure r e p r o d u c i b l e columns.


3.

Performance

DESTEFANO

Evaluation

of

LC

Columns

43

TESTING PROCEDURES


Once a d e c i s i o n i s made regarding the performance c r i t e r i a to be measured, i t then becomes necessary to design proper t e s t s . The t e s t i n g of column performance, whether i n the l a b o r a t o r y or by column manufacturers, should be c a r r i e d out w i t h the f o l l o w i n g p o i n t s i n mind: •

Since i t i s not p o s s i b l e to use t e s t s o l u t e s which would be of i n t e r e s t to a l l users of HPLC columns, t e s t mixtures should be composed of simple, s t a b l e , pure, and r e a d i l y a v a i l a b l e organic compounds.

•

Column performance i s best compared under i d e a l k i n e t i c c o n d i t i o n s . Hence, t e s t systems should be chosen which w i l l produce the best column performance (e.g., low v i s c o s i t y mobile phases, low molecular weight s o l u t e s , etc.).

•

T e s t i n g apparatus should be designed to minimize band spreading e x t e r n a l to the column (e.g., s h o r t , narrow connecting t u b i n g between the column and i n j e c t o r and d e t e c t o r , low dead-volume d e t e c t o r f l o w c e l l , e t c . ) .

•

Test procedures should be designed and d e s c r i b e d so t h a t they are r e a d i l y repeatable i n d i f f e r e n t l a b o r a t o r i e s .

SUMMARY Using a standard t e s t system, a minimum of the f o l l o w i n g s p e c i f i c a t i o n s should be determined by the user when a column i s obtained: •

Theoretical Plates

•

Peak Symmetry

•

Permeability

•

S e l e c t i v i t y (or R e t e n t i v i t y )

These column performance c r i t e r i a should be s p e c i f i e d by the manufacturer and users should measure them whenever a new column i s r e c e i v e d . These column s p e c i f i c a t i o n measurements should be kept f o r f u t u r e r e f e r e n c e and re-checked whenever i t i s suspected that the column has d e t e r i o r a t e d .



44

PESTICIDE ANALYTICAL METHODOLOGY

ABSTRACT Traditionally, HPLC column performance has been based on theoretical plate measurements. These measurements, while useful, do not provide sufficient data for complete column evaluation. In addition, when calculating theoretical plates, errors caused by peak tailing can lead to an overestimation of the resolving power of a chromatographic column. To more fully evaluate a modern, high-performance liquid chromatographic column, several performance specifications should be monitored in addition to theoretical plate measurements. These specifications include: column permeability, peak symmetry, and sample retention characteristics. A l l performance parameters should be measured under a standard set of well-documented chromatographic conditions. The factors affecting the choice of performance measures and test conditions are given. LITERATURE CITED 1.

Knox, J.; Bristow, P.; Chromatographia, 1977, 10, 279.

2.

Snyder, L.R.; Kirkland, J.J.; "Introduction to Modern Liquid Chromatography"; John Wiley and Sons, New York, 1974; pp. 35, 36, 37, 38.

3.

Kirkland, J.J.; Yau, W.W.;

Stoklosa, H.J., Dilks, Jr., C.H.;

J. Chromatogr. Sci., 1977, 15, 303. 4.

Grushka, E.; Anal. Chem., 1972, 44,

5.

Anonymous, Du Pont Instruments, L. C. Column Report - Column Performance Criteria, No. E18181. Unger, K.K.; Becker, N.; Roumeliotis, P.; J. Chromatogr. Sci., 1976, 125, 115.

6.

1733.

RECEIVED December 28, 1979.


Pesticide Analytical Methodology - American Chemical Society

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