- Email: [email protected]
Coloured Figures
la
Ib
lc
Id
Figure C I Principle of simultaneous three phase (three dimensional) circular separation in a larger IjC'Wchamber 1 a: Sample as circle line applied 1 b: Three d@Eerentphases fed with constant and equalflow 1 c: composition of mobilephase versus angle on plate I d: Readyfor quantitative and qualitative evaluation
193
Figure C 2 Simultaneous multiphase circular HFTLC chromatography dye mixture Phase A = Toluene, B = TolueneDi-isopropyl ether 50.50 vh,C = Chlorofonn
194
Figure C 3 Same dye mixture as infigure C 2 left side, but Phase A = Toluene, B = Toluenemi-isopropyl ether 5050 vh,C isopropyl ether 9020 vh and all mixtures between these three phases
=
Toluenemi-
(K) 195
figure C 4
Simultaneous multiphase circular HPTLC chromatography Phase A , B, C a l l equal = Toluene
196
figure C 5 Interesting details at trace level of polar compounds (1% ethanol in Chlorofotm) rapid changes of Rf values in ranges
(UC) 197
Figure C 6 Power of two dimensional separations: time consumption 2 x 4 minutes plate: 50 x 50 mm Phase A = Toluene, B = Chloroform
198
figure C 7 Linear chromatogram Spotting of 30 nl - 30 ng each of lipophilic dyes using a I p l Hamilton syringe in connection with a micrometer; zy-= 30 mm
10 x 10 cmplates can be used twice: 30 to 40 mm migration distance ( . = 30 - 40 mm) is suflcient for an optimization of separation in time (Halpaap)
199
Rgure C 8 Examplefor loadability of l o x lOcm HPECplates, dyespots, Toluene, 40%humidity (K) 200
figure C 9 Linear chromatogram Application of I p l = 1 pg each of lipophilic dyes in a line measuring 80 mm, using a I pl Hamilton syringe in connection with an automatic applicator. Separation power and
Rf data precision of HPT layers; dye, toluene, 40% humidity
201
Circular chromatographyis able to improve thepoor starting conditions at thesampling spot. The samples had been applied in the form of circles. TheJigure shows clearly how overloading disturbs separation power but how circular chromatography can improve the limitations of overloaded sampling (?fC) 202
figure C 11 Circular chromatogram Eccentric application of 20 nl = 20 ng each of lipophilic dyes using a I pl Hamilton syringe in connection with a micrometer;zf = 15 mm; zf = 20 mm. 0
Ideal use of separation space: 40 samples simultaneous& separated on a I0 x 10 cm plate by precise circular instrumental Hp7zC (Halpaap)
203
figure C 12 Circular chromatography on precoated H p 7 z C plates f o r nano 7zC Silica Gel 60 F254. Lipophilic dyes, mobile phase: Hexane/ChlorofomLVH~70/30, zf = 30 mm
204
Figure C I3 Circular chromatogram of Dansyl amino acids. Plate material asfigure 12. Mobile phase: Dioxanemater 97/3, zf = 30 mm, detection: UV366 nm. (Halpaap) 205
Figure C 14 Separation of cholesteryl stearate, chlormadinone acetate, cholesterone, epitestosterone, pregnandiol, corticosterone, g each. Mobile phase: chlorofotm/methanol 9713, zf = 20 mm, detection perchloric acid/methanol5/95, dipping technique 1200 C, UV366 nm. (Halpaap, E. Merck) 206
figure C 1.5 Plates as figure 14, separation of steroids as figure 14, punctiform spotting of 500, 50,30, l o x 10-9g each. Mobile phase and detection asfigure 14 (Halpaap, E. Merck)
207
Egure C 16
50 x 50 mm Hp7zC plates in addressable holder, CAMAG U-chamberfor precise qualitative 1 % at 0.5 Rf) and quantitative 2% and better reproducibility at S) analysis
(+
(+
(K) 208
Continuation of t a tfrom page 192:
The position of the peak maximum with respect to time, i.e., the migration distance
zx of the spot x, is determined by the point at which the first derivative crosses
the x-axis.
The base-line is determined according to the customary chromatographic criteria. The peak width w is given by the difference with respect to time between the two points of intersection with the base-line of the tangents through the points of inflection. The number of theoretical plates of the plate height, and the resolution between two neighboring substances, are calculated for each substance on the chromatogram from the values for the peak width w and the migration distance zx, and printed out by the computer as record of the analysis. It therefore became possible to determine the plate height for practically any number of spots on a chromatogram in less than 2 min. The approximate standard deviation for this determination of plate height for 10 bands on one plate is better than S = _+ 1.5% The corresponding manual evaluation would take many hours and yield a standard deviation of greater than k 10%of the values. An example of a computer print out is given on page 21 1.
209
210
T1 =
10.000 SEC
T2 =
210.000
SEC
220.000
SEC
time
LAUFZ E I T
T =
l a y e r t r a n s p o r t a t i o n speed migration d i s t a n c e t o t a l distance
TRANSPORT-GESCHWINDIGKEIT
U =
8.125
LAUFSTRECKE
L =
38.663
MM
GESAMTSTRECKE
S =
35.663
MM
s o l v e n t constant
FLIESSKONSTANTE
KAPPA =
5.781
PEAK NR.:
hFtf data peak base width number o f p l a t e s p l a t e number per s e c p l a t e height i n micrometer p l a t e height i n micrometer resolution
HRF-WERTE PEAK-BASISWEITE
I N MM
TRENNSTUFENZAHL TRENNSTUFENZAHLISEC
MMISEC
MM;:x2/SEC
3
2
HRF =
11.526
16.375
w =
1.738
1.681
N =
573.74
871.09
4
5
6
35.294
43.799
61.685
2.059 1252.06
2.135 1444.46
2.167 1971.86
NS =
2.060
3.959
5.691
6.565
8.963
TRENNSTUFENHOEHE I N YM
H =
53.444
35.281
24.498
21.228
15.558
TRENNSTUFENHOEHE IN YM
H50 =
12.320
AUFLOESUNG
Computerprint-out
R =
11.528 0.869
17.287 3.181
18.596 1.243
19.160 2.537
The method described made it possible to include the determination of the separating efficiency in the form of plate height and other parameters such as resolution R, Rf-value, k values and velocity coefficients K in the assessment of the quality of separated substances and also in the quality control of pre-coated plates in general.
Evaluation procedures The methods of measuring absorption and reflectance in the visible and ultraviolet spectral ranges and fluorescence, together with their optimization and limitations will be illustrated using several classes of compounds as examples. Wavelength Optimization It is customary, and in elution chromatography methods such as GC and LC in particular even essential, to evaluate substances quantitatively in the order of the k values. In TLC it is always advisable to evaluate in the direction of the solvent flow when closely neighboring or overlapping substances also exhibit slmilar absorption or fluorescence maxima. If however, the resolution between the separated substances is sufficiently large, i.e., of the order of R> 1.5, then measurement at right-angles to the solvent flow has some important advantages.
In order to demonstrate a possible optimization of the wavelength, a mixture of 7 lipophilic dyestuffs was separated on an HPTLC.
I I
1
fig. 9.3
212
L
2
3
4
i
5 6
7
Mixture of 7 lipophilic dyestuffs, 10 ng each, mobile phase benzene, and measurement at 420 nm (lower curve), 500 nm (middle curve) and 580 nm (upper curve). 1 = Ceres violet BRN 4 = Fat yellow 3G 6 = Ceres red G
2 = Ceres black G 5 = Blue VIF Organ01 7 = Ceres brown BRN
In many instances a substance exhibits its absorption maximum at a particular wavelength at which the others do not absorb. For measurements in the direction of solvent flow it is therefore always necessary to seek a compromise, since the wavelength cannot be changed during measurement with certain equipment. In the case of measurements at right-angles to the solvent flow, the optimum wavelength is set for each substance, and maximum sensitivity thus achieved. The second advantage is the possibility of referring to the background rejlectance Ro in the immediate vicinity of the spot at one point that is guaranteed to be free from substance since it lay by the side of the chromatography band. This is very important for accurate determination of the base line. The only other comparable method available for solving this problem is the doublebeam procedure in the direction of solvent flow, but without optimization of the wavelength, and that means loss of sensitivity. The third advantage is that of speed of measurement: up to 35 bands can be measured on a 10 cm wide plate with a single equipment setting (see Fig. 9.4).
Fig. 9.4
213
Section of a measurement of the blue dyestuff at right-angles to the solvent flow across 70 bands on a 20 cm wide plate. Distance separating bands: 2.5 mm Measuring conditions: A = 420 nm; slit = 1.8/0.7 mm Measuring rate: v = 30 mm/min Another example:
4I 20
a13
TIME
25
r =0,98
(r 4 , 9 9 8 1
35 46
61
91
Hy dr oc o r t is on
1 G6
121 136 145 148
250 I D 2 PW 2 0 0 0 ss
5 BL 6 0 TP 1 SP
AREA
34436 41770 42270 40491 3G607 17GS8 18725 17969 1UBOY 3188 2 21419 3 2933497
fig. 9.5
Chromatogram recorded at right-angles to the solvent flow of 20 ng and 50 ng of hydrocortisone. Integrator print-out and regression lines of the latter with and without statistically significant runaways. 214
Measuring conditions: h = 242 nm; slit 2.0/0.7 mm Measuring rate: 30 mm/min Spectra-Physics Autolab Minigrator as integrator. Hydrocortisone was determined alongside estrogens. The optimum wavelength of hydrocortisone is at 242 nm, that of the estrogens at 225 nm and 280 nm. The limit of detection of 8 x lo-'' (0.8 ng) was to be found at 1.3 x lo-'' g on measurement at right-angles to the direction of solvent flow, i.e., lower by a factor of 6. The corresponding difference can be seen clearly with the naked eye. The reason for this improvement is undoubtedly the lower background noise (see Fig. 6.6)
c
a:
4
100
)
b:
c
so
10%
20
Fig. 9.6
Comparison of the detection of 10 ng hydrocortisone (a) at right-angles to solvent flow (b) in the direction of solvent flow. 215
The fimir of detection was calculated from the equation proposed by H. Kaiser (19).
I U I-U so
= signal height =
signal height of the blank value at the same site of measurement
= limit of
detection = standard deviation of the blank values at the site of measurement
Amino acids as such are normally not detectable photometrically, and must therefore be converted chemically to absorbing or fluorescing substances before or after chromatography. Ninhydrin was used to convert amino acids into substances that absorb in the visible spectral range, and Fluescin, that is o-phthalaldehyde and mercaptoethanol in buffer solution* (17, 18) to convert such acids into substances that can be excited to produce fluorescence in the UV region. Both conversion products show similar limits of detection at about 0.1 ng, and satisfactory measurement from 1 ng upwards. Screening for phenylalanine is possible by direct application of dilute serum or plasma with a 200 nl capillary tube. In addition to the sera, phenylalanine was applied several times within pathological limits with respect to phenylketonuria, and a high, easily visible concentration attained which served to facilitate adjustment of the plate (see Fig. 9.7).
Serum
\ I
PHA
fig. 9.7
Detection of phenylalnine in serum 216
Serum
\ I
PHA
Serum
Rhodamine B, a substance which can be excited to produce intense fluorescence, was applied in concentrations between 10 ng and 20 pg (1 pg = 1 x lo-'* g) in order to ascertain whether the signal/concentration correction curve is still linear at higher amounts of sample. About 6% of all values were significant runaways which were clearly attributable to errors in application. These values were eliminated (as prescribed by statistical methods). See Figs. 9.8, 9.9,9.10 and 1.12 for the results, which are based on the calibration line passing through the origin, and exhibit a linearity with a convincing regression coefficient of 0.9997 which speaks for itself.
ID 2 pn 80 s 5
5 bL 6 0 TP
I SP
1IW F
AIIEL
Ill34
14
25 33
22560
3f105
hO44
41
5: 62
22671 45628
1.
Y48U 2 23653 3
bl 92
44 IY;
9091 2345u
I U I
IIZ
I22 I32
Fig. 9.8 Rhodamine B
05/40
10531 ?A593 41330
142
152 I l U
I 16
534 2 5871 3 3832601
1
A
I PW
50 SS
I
a 0
. I
[of
-Ah?'
AREA
23
123U
5J GJ
13
61 94
I03 llJ 123 133
Rhodamine B
I SP
Tlhr
34 43
Fig. 9.9
p&
143
IS3
163
I83
sr
ID ID I P1 50 SS 5 aL 6 0 TP
3418 6831
Ill? 2582 6461 1381 2d21
6455
1043 2Y99 620b 1213
2934 6092
12G 1 533357 '
*I
h
I,
h 217
57
1503 2 19b 3
59 67 72 7b
6a5
131 142
-0 0
91
95 106 112
117
l5u 2
31 2
--
b59_ 3 3RG 2
___
lAg& 3
681
127 - 6 1 9 133 137 I 1U 8 5 151
153 157 I L7
161
fig. 9.10 Rhodamine B
56 2 216-3 . 2 48 2 123 2
195 3
z -T i
116
~
e
-
- ?
i
20 10
5 2 1
.. I
#
.A
fig. 9.11
Regression line and correlation coefficient for the values from Figs. 9.8-9.10 (see Table 1)
218
Table 1 Rhodamine B
1 10 ng
Approximate recurring standard deviation measured in dirction of flow in O/o
5 ng
2 ng
n' r ' -
Approximate standard deviation measured at right angles to flow in O/o Rhodamine B Approximate standard deviation measured at right angles to flow in OIo
r a b
1 0 0 pg 50 PR 2 0
= gradient of
regression line = coordinate section of regression line
b'
n r
a
b
4,383
+0,07
6 0,997
4,386
+0,07
0,9998
4,502
+0,29
6 0,998
4,507
+0,27
16 0 , 9 9 8 8
4,183
-0,91
6 0,985
4,027
-0,18
pg
+3,3% +5,0%+15,8%
= linear correlation coefficient
a'
0,9999
(at 1.OOO there is a theoretical calibration line which in practice does not exist).
n' gives the number of individual measurements for one evaluation, and includes all values on the plate. n includes the neighboring values or two values in each case per concentration. At 2 ng and 20 pg the dosage was 20 nl, and the approximate standard deviation is therefore comparatively large.
It can be seen from Table 1 that there is no difference in reproducibility and accuracy of the quantitative results of the analysis between the values in and at right-angles to the direction of flow, apart from the considerably lowered limit of detection. There is, however, a considerable saving in time at rightangles to the direction of flow, since it is only necessary to adjust the band measured once, instead of 14 times as in the other case. For Rhodamine B a limit of detection N =6 x g was found. The aflatoxins B1, B2, G1 and G2 were separated on an HPTLC plate by twofold development with chloroform-acetone 9O:lO. Amounts of 200 pg, 500 pg and lo00 pg were applied to determine the calibration line. Twenty-four bands could be accommodated on a 10 cm wide plate, which made it possible to perform an eightfold determination for each concentration. Application of the substances, chromatography, measurement and evaluations took altogether about 1 hour working time. Three of the 24 bands are represented in Fig. 9.12. The limit of detection for the aflatoxins is at 10 pg. The regression line passes through the origin, and its correlation coefficients are in all cases better than r = 0.9987. In this range therefore, there is a linear relationship between fluorescence signal and amount of substance.
220
200 pg
500 P9
fig.9.12
Section from evaluation of a plate accommodating a total .of 24 bands aflatoxins. Excitation wavelength is 366 nm Measurement wavelength is 460 nm Slit dimension = 3.W0.7 mm Measuring rate 30 mm/min Chromatographic conditions: HPTLC plate, mobile phase chloroform/acetone 9O:lO v/v, twice, in each case 70 mm high; N-chamber; chamber saturation. 22 1
Table 2 summarizes the aflatoxin data
Substance Amount in pg
rel. approximate standard deviation
n‘
r’
a‘
22
0,9987
1,74 3.27
b’
b
n
r
a
+0,04
6
0,997
1,78
-0,27
+0,65
6
0,998
3,39
+0,13 +0,38
A f l a t o x i n B1
1000 500 200 pg + 2 , 5 % +3,0% & 1 2 , 5 %
A f l a t o x i n B2
+1,5% t3,2%
+3,5%
A f l a t o x i n G1
+3,6% +4,1%
+9,1%
24
0,9998
1,286+0,27
6
0,998
1,28
Aflatoxin G2
+2,4% +4,5%
+7,0%
23
0,9998
1,795
6
0,992
1,756+0,13
24
0,9995
The above data underline the accuracy with which nanogram analysis may be camed out on HFTLC plates, since the example reported has nothing to do with “dyestuff mixtures in the ranges”.
-0,29
Regression Line “Aflatoxins” and Example of Structure.
0 0
mbs
30
II
II
r/ Aflatoxin
GI
20
10
200
500
1000
pg
223
The standard deviations of the individual values for lo00 pg, corresponding to 100 nanoliters volume of sample, were between 1.5 and f 3.6% of the value, and for 200 pg, corresponding to 20 nanoliters volume of sample, between f3.5 and f 12.5%of the value.
*
It was to be expected that with 24 measurements or 8 values per concentration the correlation coefficient would come out better than with measurement of 6 neighboring bands or the equivalent 2 values per concentration. Gradient and coordinate section - i.e., passage through the origin - remained practically unchanged, however, so that for determination of the correction curve two determinations in each case from two or three concentrations affords adequate accuracy. The HPTLC plate could then accommodate 18 to 20 bands for the analytical determination itself.
Our interest in the future will continue to be directed towards simplification of analytical procedures, e.g., by “cleanup” directly on the HPTLC plate, and also towards improvement of the sensitivity of detection, possibly by chemical reactions on the plate. It is not impossible that in the near future the limits of detection of some classes of substances will be pushed into the femtogram range, as currently done with radio-labelled substances.
224
Ib
lc
Id
Figure C I Principle of simultaneous three phase (three dimensional) circular separation in a larger IjC'Wchamber 1 a: Sample as circle line applied 1 b: Three d@Eerentphases fed with constant and equalflow 1 c: composition of mobilephase versus angle on plate I d: Readyfor quantitative and qualitative evaluation
193
Figure C 2 Simultaneous multiphase circular HFTLC chromatography dye mixture Phase A = Toluene, B = TolueneDi-isopropyl ether 50.50 vh,C = Chlorofonn
194
Figure C 3 Same dye mixture as infigure C 2 left side, but Phase A = Toluene, B = Toluenemi-isopropyl ether 5050 vh,C isopropyl ether 9020 vh and all mixtures between these three phases
=
Toluenemi-
(K) 195
figure C 4
Simultaneous multiphase circular HPTLC chromatography Phase A , B, C a l l equal = Toluene
196
figure C 5 Interesting details at trace level of polar compounds (1% ethanol in Chlorofotm) rapid changes of Rf values in ranges
(UC) 197
Figure C 6 Power of two dimensional separations: time consumption 2 x 4 minutes plate: 50 x 50 mm Phase A = Toluene, B = Chloroform
198
figure C 7 Linear chromatogram Spotting of 30 nl - 30 ng each of lipophilic dyes using a I p l Hamilton syringe in connection with a micrometer; zy-= 30 mm
10 x 10 cmplates can be used twice: 30 to 40 mm migration distance ( . = 30 - 40 mm) is suflcient for an optimization of separation in time (Halpaap)
199
Rgure C 8 Examplefor loadability of l o x lOcm HPECplates, dyespots, Toluene, 40%humidity (K) 200
figure C 9 Linear chromatogram Application of I p l = 1 pg each of lipophilic dyes in a line measuring 80 mm, using a I pl Hamilton syringe in connection with an automatic applicator. Separation power and
Rf data precision of HPT layers; dye, toluene, 40% humidity
201
Circular chromatographyis able to improve thepoor starting conditions at thesampling spot. The samples had been applied in the form of circles. TheJigure shows clearly how overloading disturbs separation power but how circular chromatography can improve the limitations of overloaded sampling (?fC) 202
figure C 11 Circular chromatogram Eccentric application of 20 nl = 20 ng each of lipophilic dyes using a I pl Hamilton syringe in connection with a micrometer;zf = 15 mm; zf = 20 mm. 0
Ideal use of separation space: 40 samples simultaneous& separated on a I0 x 10 cm plate by precise circular instrumental Hp7zC (Halpaap)
203
figure C 12 Circular chromatography on precoated H p 7 z C plates f o r nano 7zC Silica Gel 60 F254. Lipophilic dyes, mobile phase: Hexane/ChlorofomLVH~70/30, zf = 30 mm
204
Figure C I3 Circular chromatogram of Dansyl amino acids. Plate material asfigure 12. Mobile phase: Dioxanemater 97/3, zf = 30 mm, detection: UV366 nm. (Halpaap) 205
Figure C 14 Separation of cholesteryl stearate, chlormadinone acetate, cholesterone, epitestosterone, pregnandiol, corticosterone, g each. Mobile phase: chlorofotm/methanol 9713, zf = 20 mm, detection perchloric acid/methanol5/95, dipping technique 1200 C, UV366 nm. (Halpaap, E. Merck) 206
figure C 1.5 Plates as figure 14, separation of steroids as figure 14, punctiform spotting of 500, 50,30, l o x 10-9g each. Mobile phase and detection asfigure 14 (Halpaap, E. Merck)
207
Egure C 16
50 x 50 mm Hp7zC plates in addressable holder, CAMAG U-chamberfor precise qualitative 1 % at 0.5 Rf) and quantitative 2% and better reproducibility at S) analysis
(+
(+
(K) 208
Continuation of t a tfrom page 192:
The position of the peak maximum with respect to time, i.e., the migration distance
zx of the spot x, is determined by the point at which the first derivative crosses
the x-axis.
The base-line is determined according to the customary chromatographic criteria. The peak width w is given by the difference with respect to time between the two points of intersection with the base-line of the tangents through the points of inflection. The number of theoretical plates of the plate height, and the resolution between two neighboring substances, are calculated for each substance on the chromatogram from the values for the peak width w and the migration distance zx, and printed out by the computer as record of the analysis. It therefore became possible to determine the plate height for practically any number of spots on a chromatogram in less than 2 min. The approximate standard deviation for this determination of plate height for 10 bands on one plate is better than S = _+ 1.5% The corresponding manual evaluation would take many hours and yield a standard deviation of greater than k 10%of the values. An example of a computer print out is given on page 21 1.
209
210
T1 =
10.000 SEC
T2 =
210.000
SEC
220.000
SEC
time
LAUFZ E I T
T =
l a y e r t r a n s p o r t a t i o n speed migration d i s t a n c e t o t a l distance
TRANSPORT-GESCHWINDIGKEIT
U =
8.125
LAUFSTRECKE
L =
38.663
MM
GESAMTSTRECKE
S =
35.663
MM
s o l v e n t constant
FLIESSKONSTANTE
KAPPA =
5.781
PEAK NR.:
hFtf data peak base width number o f p l a t e s p l a t e number per s e c p l a t e height i n micrometer p l a t e height i n micrometer resolution
HRF-WERTE PEAK-BASISWEITE
I N MM
TRENNSTUFENZAHL TRENNSTUFENZAHLISEC
MMISEC
MM;:x2/SEC
3
2
HRF =
11.526
16.375
w =
1.738
1.681
N =
573.74
871.09
4
5
6
35.294
43.799
61.685
2.059 1252.06
2.135 1444.46
2.167 1971.86
NS =
2.060
3.959
5.691
6.565
8.963
TRENNSTUFENHOEHE I N YM
H =
53.444
35.281
24.498
21.228
15.558
TRENNSTUFENHOEHE IN YM
H50 =
12.320
AUFLOESUNG
Computerprint-out
R =
11.528 0.869
17.287 3.181
18.596 1.243
19.160 2.537
The method described made it possible to include the determination of the separating efficiency in the form of plate height and other parameters such as resolution R, Rf-value, k values and velocity coefficients K in the assessment of the quality of separated substances and also in the quality control of pre-coated plates in general.
Evaluation procedures The methods of measuring absorption and reflectance in the visible and ultraviolet spectral ranges and fluorescence, together with their optimization and limitations will be illustrated using several classes of compounds as examples. Wavelength Optimization It is customary, and in elution chromatography methods such as GC and LC in particular even essential, to evaluate substances quantitatively in the order of the k values. In TLC it is always advisable to evaluate in the direction of the solvent flow when closely neighboring or overlapping substances also exhibit slmilar absorption or fluorescence maxima. If however, the resolution between the separated substances is sufficiently large, i.e., of the order of R> 1.5, then measurement at right-angles to the solvent flow has some important advantages.
In order to demonstrate a possible optimization of the wavelength, a mixture of 7 lipophilic dyestuffs was separated on an HPTLC.
I I
1
fig. 9.3
212
L
2
3
4
i
5 6
7
Mixture of 7 lipophilic dyestuffs, 10 ng each, mobile phase benzene, and measurement at 420 nm (lower curve), 500 nm (middle curve) and 580 nm (upper curve). 1 = Ceres violet BRN 4 = Fat yellow 3G 6 = Ceres red G
2 = Ceres black G 5 = Blue VIF Organ01 7 = Ceres brown BRN
In many instances a substance exhibits its absorption maximum at a particular wavelength at which the others do not absorb. For measurements in the direction of solvent flow it is therefore always necessary to seek a compromise, since the wavelength cannot be changed during measurement with certain equipment. In the case of measurements at right-angles to the solvent flow, the optimum wavelength is set for each substance, and maximum sensitivity thus achieved. The second advantage is the possibility of referring to the background rejlectance Ro in the immediate vicinity of the spot at one point that is guaranteed to be free from substance since it lay by the side of the chromatography band. This is very important for accurate determination of the base line. The only other comparable method available for solving this problem is the doublebeam procedure in the direction of solvent flow, but without optimization of the wavelength, and that means loss of sensitivity. The third advantage is that of speed of measurement: up to 35 bands can be measured on a 10 cm wide plate with a single equipment setting (see Fig. 9.4).
Fig. 9.4
213
Section of a measurement of the blue dyestuff at right-angles to the solvent flow across 70 bands on a 20 cm wide plate. Distance separating bands: 2.5 mm Measuring conditions: A = 420 nm; slit = 1.8/0.7 mm Measuring rate: v = 30 mm/min Another example:
4I 20
a13
TIME
25
r =0,98
(r 4 , 9 9 8 1
35 46
61
91
Hy dr oc o r t is on
1 G6
121 136 145 148
250 I D 2 PW 2 0 0 0 ss
5 BL 6 0 TP 1 SP
AREA
34436 41770 42270 40491 3G607 17GS8 18725 17969 1UBOY 3188 2 21419 3 2933497
fig. 9.5
Chromatogram recorded at right-angles to the solvent flow of 20 ng and 50 ng of hydrocortisone. Integrator print-out and regression lines of the latter with and without statistically significant runaways. 214
Measuring conditions: h = 242 nm; slit 2.0/0.7 mm Measuring rate: 30 mm/min Spectra-Physics Autolab Minigrator as integrator. Hydrocortisone was determined alongside estrogens. The optimum wavelength of hydrocortisone is at 242 nm, that of the estrogens at 225 nm and 280 nm. The limit of detection of 8 x lo-'' (0.8 ng) was to be found at 1.3 x lo-'' g on measurement at right-angles to the direction of solvent flow, i.e., lower by a factor of 6. The corresponding difference can be seen clearly with the naked eye. The reason for this improvement is undoubtedly the lower background noise (see Fig. 6.6)
c
a:
4
100
)
b:
c
so
10%
20
Fig. 9.6
Comparison of the detection of 10 ng hydrocortisone (a) at right-angles to solvent flow (b) in the direction of solvent flow. 215
The fimir of detection was calculated from the equation proposed by H. Kaiser (19).
I U I-U so
= signal height =
signal height of the blank value at the same site of measurement
= limit of
detection = standard deviation of the blank values at the site of measurement
Amino acids as such are normally not detectable photometrically, and must therefore be converted chemically to absorbing or fluorescing substances before or after chromatography. Ninhydrin was used to convert amino acids into substances that absorb in the visible spectral range, and Fluescin, that is o-phthalaldehyde and mercaptoethanol in buffer solution* (17, 18) to convert such acids into substances that can be excited to produce fluorescence in the UV region. Both conversion products show similar limits of detection at about 0.1 ng, and satisfactory measurement from 1 ng upwards. Screening for phenylalanine is possible by direct application of dilute serum or plasma with a 200 nl capillary tube. In addition to the sera, phenylalanine was applied several times within pathological limits with respect to phenylketonuria, and a high, easily visible concentration attained which served to facilitate adjustment of the plate (see Fig. 9.7).
Serum
\ I
PHA
fig. 9.7
Detection of phenylalnine in serum 216
Serum
\ I
PHA
Serum
Rhodamine B, a substance which can be excited to produce intense fluorescence, was applied in concentrations between 10 ng and 20 pg (1 pg = 1 x lo-'* g) in order to ascertain whether the signal/concentration correction curve is still linear at higher amounts of sample. About 6% of all values were significant runaways which were clearly attributable to errors in application. These values were eliminated (as prescribed by statistical methods). See Figs. 9.8, 9.9,9.10 and 1.12 for the results, which are based on the calibration line passing through the origin, and exhibit a linearity with a convincing regression coefficient of 0.9997 which speaks for itself.
ID 2 pn 80 s 5
5 bL 6 0 TP
I SP
1IW F
AIIEL
Ill34
14
25 33
22560
3f105
hO44
41
5: 62
22671 45628
1.
Y48U 2 23653 3
bl 92
44 IY;
9091 2345u
I U I
IIZ
I22 I32
Fig. 9.8 Rhodamine B
05/40
10531 ?A593 41330
142
152 I l U
I 16
534 2 5871 3 3832601
1
A
I PW
50 SS
I
a 0
. I
[of
-Ah?'
AREA
23
123U
5J GJ
13
61 94
I03 llJ 123 133
Rhodamine B
I SP
Tlhr
34 43
Fig. 9.9
p&
143
IS3
163
I83
sr
ID ID I P1 50 SS 5 aL 6 0 TP
3418 6831
Ill? 2582 6461 1381 2d21
6455
1043 2Y99 620b 1213
2934 6092
12G 1 533357 '
*I
h
I,
h 217
57
1503 2 19b 3
59 67 72 7b
6a5
131 142
-0 0
91
95 106 112
117
l5u 2
31 2
--
b59_ 3 3RG 2
___
lAg& 3
681
127 - 6 1 9 133 137 I 1U 8 5 151
153 157 I L7
161
fig. 9.10 Rhodamine B
56 2 216-3 . 2 48 2 123 2
195 3
z -T i
116
~
e
-
- ?
i
20 10
5 2 1
.. I
#
.A
fig. 9.11
Regression line and correlation coefficient for the values from Figs. 9.8-9.10 (see Table 1)
218
Table 1 Rhodamine B
1 10 ng
Approximate recurring standard deviation measured in dirction of flow in O/o
5 ng
2 ng
n' r ' -
Approximate standard deviation measured at right angles to flow in O/o Rhodamine B Approximate standard deviation measured at right angles to flow in OIo
r a b
1 0 0 pg 50 PR 2 0
= gradient of
regression line = coordinate section of regression line
b'
n r
a
b
4,383
+0,07
6 0,997
4,386
+0,07
0,9998
4,502
+0,29
6 0,998
4,507
+0,27
16 0 , 9 9 8 8
4,183
-0,91
6 0,985
4,027
-0,18
pg
+3,3% +5,0%+15,8%
= linear correlation coefficient
a'
0,9999
(at 1.OOO there is a theoretical calibration line which in practice does not exist).
n' gives the number of individual measurements for one evaluation, and includes all values on the plate. n includes the neighboring values or two values in each case per concentration. At 2 ng and 20 pg the dosage was 20 nl, and the approximate standard deviation is therefore comparatively large.
It can be seen from Table 1 that there is no difference in reproducibility and accuracy of the quantitative results of the analysis between the values in and at right-angles to the direction of flow, apart from the considerably lowered limit of detection. There is, however, a considerable saving in time at rightangles to the direction of flow, since it is only necessary to adjust the band measured once, instead of 14 times as in the other case. For Rhodamine B a limit of detection N =6 x g was found. The aflatoxins B1, B2, G1 and G2 were separated on an HPTLC plate by twofold development with chloroform-acetone 9O:lO. Amounts of 200 pg, 500 pg and lo00 pg were applied to determine the calibration line. Twenty-four bands could be accommodated on a 10 cm wide plate, which made it possible to perform an eightfold determination for each concentration. Application of the substances, chromatography, measurement and evaluations took altogether about 1 hour working time. Three of the 24 bands are represented in Fig. 9.12. The limit of detection for the aflatoxins is at 10 pg. The regression line passes through the origin, and its correlation coefficients are in all cases better than r = 0.9987. In this range therefore, there is a linear relationship between fluorescence signal and amount of substance.
220
200 pg
500 P9
fig.9.12
Section from evaluation of a plate accommodating a total .of 24 bands aflatoxins. Excitation wavelength is 366 nm Measurement wavelength is 460 nm Slit dimension = 3.W0.7 mm Measuring rate 30 mm/min Chromatographic conditions: HPTLC plate, mobile phase chloroform/acetone 9O:lO v/v, twice, in each case 70 mm high; N-chamber; chamber saturation. 22 1
Table 2 summarizes the aflatoxin data
Substance Amount in pg
rel. approximate standard deviation
n‘
r’
a‘
22
0,9987
1,74 3.27
b’
b
n
r
a
+0,04
6
0,997
1,78
-0,27
+0,65
6
0,998
3,39
+0,13 +0,38
A f l a t o x i n B1
1000 500 200 pg + 2 , 5 % +3,0% & 1 2 , 5 %
A f l a t o x i n B2
+1,5% t3,2%
+3,5%
A f l a t o x i n G1
+3,6% +4,1%
+9,1%
24
0,9998
1,286+0,27
6
0,998
1,28
Aflatoxin G2
+2,4% +4,5%
+7,0%
23
0,9998
1,795
6
0,992
1,756+0,13
24
0,9995
The above data underline the accuracy with which nanogram analysis may be camed out on HFTLC plates, since the example reported has nothing to do with “dyestuff mixtures in the ranges”.
-0,29
Regression Line “Aflatoxins” and Example of Structure.
0 0
mbs
30
II
II
r/ Aflatoxin
GI
20
10
200
500
1000
pg
223
The standard deviations of the individual values for lo00 pg, corresponding to 100 nanoliters volume of sample, were between 1.5 and f 3.6% of the value, and for 200 pg, corresponding to 20 nanoliters volume of sample, between f3.5 and f 12.5%of the value.
*
It was to be expected that with 24 measurements or 8 values per concentration the correlation coefficient would come out better than with measurement of 6 neighboring bands or the equivalent 2 values per concentration. Gradient and coordinate section - i.e., passage through the origin - remained practically unchanged, however, so that for determination of the correction curve two determinations in each case from two or three concentrations affords adequate accuracy. The HPTLC plate could then accommodate 18 to 20 bands for the analytical determination itself.
Our interest in the future will continue to be directed towards simplification of analytical procedures, e.g., by “cleanup” directly on the HPTLC plate, and also towards improvement of the sensitivity of detection, possibly by chemical reactions on the plate. It is not impossible that in the near future the limits of detection of some classes of substances will be pushed into the femtogram range, as currently done with radio-labelled substances.
224